Review Dissecting lipid raft facilitated cell signaling ...€¦ · Review Dissecting lipid raft...

Available online at www.sciencedirect.com

Biochimica et Biophysica Acta 1785 (2008) 182–206www.elsevier.com/locate/bbacan

Review

Dissecting lipid raft facilitated cell signaling pathways in cancer

Samir Kumar Patra ⁎

Cancer Epigenetics Research, Kalyani (B–7/183), Nadia, West Bengal, India–741235

Received 4 October 2007; received in revised form 24 November 2007; accepted 29 November 2007Available online 5 December 2007

Abstract

Cancer is one of the most devastating disorders in our lives. Higher rate of proliferation than death of cells is one of the essential factors fordevelopment of cancer. The dynamicity of cell membrane plays some vital roles in cell survival and cell death, including protection, endocytosis,signaling, and increases in mechanical stability during cell division, as well as decrease of shear forces during separation of two cells after division,and cell separation from tissues for cancer metastasis. Within the membrane, there are specialized domains, known as lipid rafts. A raft can coordinatevarious signaling pathways. Recent data on the proteomics of lipid rafts/caveolae have highlighted the enigmatic role of various signaling proteins incancer development. Analysis of these data of raft proteome from various tumors, cancer tissues, and cell lines cultured without and with therapeuticagents, as well as from model rafts revealed that there may be two subsets of raft assemblage in cell membrane. One subset of raft is enriched withcholesterol–sphingomyeline–ganglioside–cav-1/Src/EGFR (hereafter, “chol-raft”) that is involved in normal cell signaling, and when dysregulatedpromotes cell transformation and tumor progression; another subset of raft is enriched with ceramide–sphingomyeline–ganglioside–FAS/Ezrin(hereafter, “cer-raft”) that generally promotes apoptosis. In view of this, and to focus insight into the cancer cell physiology caused by the lipid raftsmediated signals and their receptors, and the downstream transmitters, either proliferative (for example, EGF and EGFR) or death-inducing(for example, FASL and FAS), and the precise roles of some therapeutic drugs and endogenous acid sphingomylenase in this scenario in in situtransformation of “chol-raft” into “cer-raft” are summarized and discussed in this contribution.© 2007 Elsevier B.V. All rights reserved.

Keywords: Cancer; Catenin; Caveolin-1; CD44; Ceramide; EGFR; Cholesterol; E-cadherin; Ezrin; FAS/CD95; FASL; Focal adhesion kinase; H-ras; Integrin; Lipidrafts; Matrix metalloproteinases (MMPs); Proteomics; Signal transduction; Sphingomyelin; uPA; uPAR; MAP kinase

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1831.1. The flora and fauna of lipid rafts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

2. Lipid rafts/caveolae signaling and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852.1. Raft and caveolin-1 signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1862.2. Tumor suppression and metastasis promotion. The cav-1, CD44, E-cadherin paradoxes . . . . . . . . . . . . . . . . . . . 186

3. Raft and epidermal growth factor receptor (EGFR) signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1884. Nuclear factor kappa B signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1905. Rafts in MAP kinase, Ras, and activator protein 1 (AP1) signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Abbreviations:Acid sphingomyelinase, ASMase; Activator protein-1, AP-1; Caveolin-1, cav-1; Ceramide, Cer; Cholesterol, Chol; Extracellular matrix, ECM;E-cadherin,E-cad; Endoplasmic reticulum, ER; Epidermal growth factor, EGF; EGF receptor, EGFR; Extracellular signal-regulated kinase, ERK; FAS antigen, FAS; FAS associated deathdomain, FADD; FAS ligand, FASL, Death-inducing signaling complex, DISC; Focal adhesion kinase, FAK; Glycosyl phosphatidyl inositol, GPI; Insulin like growth factor,IGF;Matrixmetalloproteinases,MMPs; Mitogen activated protein kinase,MAPK;MAP/ERK kinase 1/2, MEK1/2; Nuclear factor-kB, NF-kB; Phosphoinositide 3-kinase,PI3K; Plasma membranes, PM; Receptor tyrosine kinases, RTKs; Retinoic acid, RA; RA receptor, RAR; Sentinel lymph nodes, SLN; Sphingomyelin, SM;Thrombospondine 2, THBS2; Urokinase type plasminogen activator (uPA); uPA receptor (uPAR)⁎ Corresponding author. Tel.: +91 9432060602; fax: +91 3325828460.E-mail address: [email protected].

0304-419X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbcan.2007.11.002

mailto:[email protected]

http://dx.doi.org/10.1016/j.bbcan.2007.11.002

183S.K. Patra / Biochimica et Biophysica Acta 1785 (2008) 182–206

6. Raft and insulin like growth factor mediated signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1907. Extracellular matrix and raft signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1908. Rafts in MMPs and uPAR signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1919. Rafts in integrin and focal adhesion kinase mediated signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19110. Raft signaling and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19211. FAS and raft mediated FAS signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19212. Rafts and ezrin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19413. Acid sphingomyelinase and raft signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19514. Discussion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

14.1. Tumour suppression and metastasis promotion duality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

1. Introduction

Maintenance of balance between cell proliferation and celldeath is the main key of normal development. Cancer cells havehigher rate of proliferation than death. Usually, deregulated cellcycle is the cause of such impairment. Deregulation of cell cycle iscaused by aberrant signaling. The dynamicity of membranes andflip-flop flexibility of lipids within cell membrane play some vitalroles in cells life and death. Plasma membrane gives a protectionto cytoplasmic ingredients and organelles, it perform endocytosisand signaling, and increases the mechanical stability of cellsduring division, as well as the flexibility of lipids within themembrane causes decrease of shear forces during cell separation(for example, separation of individual cells from the host tumorduring cancer metastasis). Within the membrane, there arespecialized domains, known as lipid rafts. Many proteins ofreceptor tyrosine kinases (RTK) family members, includingepidermal growth factor receptor (EGFR), and other proteins,including caveolin-1, CD44, uPAR, H-Ras, integrins and cateninshave been implicated to various cellular functions, includingstability and signaling. Some of these proteins precisely exhibittheir function through lipid rafts, either structurally or function-ally, or both, in immune signaling, angiogenesis, cell polarity andcancer progression. Function of proteins like, FAS and FASLvirtually remain inert, which in turn facilitate tumor developmentby reduced rate of apoptosis. Also, impaired function of FAS andFASL results in tumor development, immune disorders and otherdiseases, including diabetes and Parkinson's disease. Investiga-tions on the molecular mechanisms of cell transformation anddevelopment of various cancers, including breast, lung, prostate,gliomas and multiple sarcomas are given immense importance,since cancer is one of the major threats in our life. We have beenworking for years on molecular and epigenetic regulation ofcancer, including lipid rafts, DNAmethylation, and lipid rafts andcancer metastasis [1]. In this contribution, I shall discuss someimportant signaling events leading to cell transformation andcancer progression, which otherwise depend predominantly onlipid rafts. A handfull collective knowledge of lipid rafts and raft-assisted signaling pathways would help us to choose strategies forprevention, cure and better management of cancers using natu-ral compounds, synthetic inhibitors, radiation or other forms oftherapies.

1.1. The flora and fauna of lipid rafts

Lipid/membrane rafts are small (10–200 nm), heterogeneous,highly dynamic, sterol- and sphingolipid-enriched domains thatcompartmentalize cellular processes. Caveolae, a subclass ofrafts, are characterized by flask-like invaginations of the plasmamembrane that are distinguished from bulk lipid rafts by thepresence of caveolin-1 (cav-1). Hence, lipid rafts/caveolae arespecialized molecular assemblages of sphingolipids and choles-terol, orchestrated by proteins and gangliosides that are knownprincipally for their pivotal role in trans-cytosis, sorting ofsphingolipids and cholesterol in the cell, and as platforms toconcentrate receptors and assembling the signal transductionmachinery; but their ability to influence the actin cytoskeleton,cell polarity, angiogenesis, membrane fusion is probably just assignificant [1–20]. Fig. 1. shows a schematic view of lipid rafts,and caveolae like compositions. The lower half of the Fig. 1depicts a typical composition of a cell death associated raft clus-tering enriched with ceramide (will be discussed below). All thecomponents (lipids and proteins) presented in Fig. 1 are notavailable in the same type of raft. The raft composition largelydepends on what fraction of lipids in the cytoplasmic leaflet formrafts in living cells and the type of cellular response after receivingsignal/stimuli [2–31]. All tumor cells shed plasma membranesenriched in sphingomyelin (SM), cholesterol and gangliosides tocounter possibly against hosts immune responses and keepthemselves free from destruction by immune system (reviewed inref. [1], see also [28–32]).

The lipid raft proteomics is the study of all the proteins thatuse, and most importantly need raft assemblage for their properfunctioning, certainly expressed by a given cell, tissue or or-ganism at a given time and under specific conditions. Some ofthose proteins are well illustrated in the case of signaling inhematopoietic cells, including T-cells and B-cells, in a variety ofcancer cells and to some extent inmodel rafts [1–17,19–26,33–53].The binding of actin is an important example of interaction of raftcomponents with cytoplasmic proteins, which implies raftmediated signaling, cell surface organization and a role for raftsin mechanical properties of cell membranes. Actin forms protein-chains such as Cadherin–Catenin–Actin, CD44–(Ezrin, Radixin,Moesin; ERM)–Actin, and some others depending on tissue andcell types where catenin and ERM-like proteins constitute a

Fig. 1. Schematic depiction of a lipid raft. Note that, all the components shown here are not present in the same type of raft. For various types of rafts with distinctcomposition see references [1–6, 8–10, 13–17, 25–27]. Lower half shows typical composition of a cell death associated raft clustering enriched with ceramide whencells and tumors are exposed to radiation or challenged with therapeutic compounds. The coupling between outer and cytoplasmic leaflets is hypothetical. See alsoreferences [24, 153, 154, 224–226].

184 S.K. Patra / Biochimica et Biophysica Acta 1785 (2008) 182–206

molecular bridge. Table 1 summarizes the lipid components, andTable 2 shows a few protein components of rafts as identified bybiophysical, biochemical, and immuno-localization methods. Allraft components cited in Table 2 are authentic and supported from

the works of at least four separate laboratories, and furtherconfirmed by unbiased proteomics of lipid rafts [1,4,34–36,40–45]. The proteomics approach yielded, quantified and validatedaround 250 proteins as authentic raft proteins [4,35–37],

Table 1Lipid components of rafts — their function and regulation mechanism

Component lipids Major function/(abundance in cancer withcomparison to normal tissues: very high ↑↑,high↑, low ↓, or cancer stage specific↑↓)

Mechanism of control/regulation References

Cholesterol (Chol) Spacer between the hydrocarbon chains,H-bonding with surface water andsphingomyelin, implicated for signaltransduction, precursor for steroidbiosynthesis/(Yes ↑↑)

Biosynthesis, efflux from and influxinto cells by transport of lipoproteinscholesterol, endocytic recycling.

[1,5,8,11–17,46,52,71,119,235–238] and references therein

Sphingomyelin (SM) Maintainance of bilayer structure,macrodomain formation, signaling/(Yes ↑)

Biosynthesis, and endocytic recycling [1,5,8,11–17,22–24,46,31,119,224–227,244]

Glycosphingolipids (e.g., GM1) Various signaling andImmuno-protection/(Yes ↑)

Biosynthesis, and endocytic recycling [1,5,8,11–17,28–31,49]

Lysophosphatidic acid (LPA) Signal transduction/(Yes ↑) Biosynthesis [1,8,224,227,229]PIP2 Signal transduction/(Yes ↑) Biosynthesis [8,224,229]Ceramide (Cer) Signal transduction (↑↓)/FasL

mediated apoptosis (Yes ↑↑)Biosynthesis, Sphingomyelin breakdown by ASMase

[19,20,22–24,119,224–227,229]


excluding the contaminations of non-raft detergent-resistant/insoluble membrane proteins [17,38,39]. From such a large set oftrue raft proteins, I have picked some structural as well assignaling molecules, including cav-1, CD44, EGFR, Ras, uPAR,MMPs, and FAS, Ezrin and a few related proteins for discussionin the following sections.

Manipulation of available data on lipid rafts by proteomicsapproach for various cancer cells and of model rafts havecompelled to suggest that there may be two subsets of raftassemblage in cell membrane. One subset of raft is enriched with

Table 2Examples of protein components of lipid rafts

Marked fluctuationsin the abundance ofraft proteins in cancers

Swiss Prot/OMIM References

Caveolin-1 Q03135/601047 [1,5,8,36,46,47,51,70]β-Actin P02570 [1,5,8]uPAR, Q03405/173391 Reviewed in ref.

[1,129–131,36,179]uPA 191840 Reviewed in ref. [129],

and [176–178]β-Catenin 116806 [1,88–95]α-Catenin P26232 [36,88–95]CD44 107269 [1,49,76,77]FAS/CD95 134637 [24,153,154,157,159–170,

199,224–228]Ezrin P26038/123900 [1,4,36,143,210–212,

153,154,225]Ras (R-Ras2, TC21) P17082 [1,8,15,122]Ras-related (Rap2) P17964 [5,8,36]Ras GAP-like IQGAP P46940 [36]Integrin, beta 1 P05556 [1,36,130,131,133]MMP-9 120361 [1,130,133,134,249]MMP-2 120360 [1,130,137–141,248]Receptor tyrosine kinases,

EGFRO15146/131550 [8,107,111–119]

Serine/Threonine kinase 35 IPI00104087.3 [5,8,36]

The raft protein itself, or one of its partners (which essentially have molecularinteraction) in the signaling cascades are presented.

cholesterol–sphingomyeline–ganglioside (hereafter, “chol-raft”)containing proteins mainly Caveolins, CD44, andmembers of theRTKs family. “Chol-rafts” are responsible for cellular home-ostasis, but when normal cellular signaling is dysregulated “chol-rafts” promote cell transformation, tumor progression, angiogen-esis and metastasis. Another subset of raft is enriched withceramide–sphingomyeline (hereafter, “cer-raft”) containingmainly, FAS, FASL, and themembers of death-inducing signalingcomplex (DISC). “Cer-rafts” promote apoptosis. Ezrin likemolecules function as molecular bridge in both types, the “chol-rafts” and the “cer-rafts”. Small rafts can sometimes be stabilizedto form larger platforms through clustering of proteins, and lipid–protein–lipid raft (LPLR) reordering in living cells by elevatedcholesterol induced coalescence of rafts. This reordering of “chol-raft”might serve to sequester proteins namely, CD44, EGFR, Rasand stimulate “start” signals to oncogenic pathways. When cellsand tumors are exposed to radiation or challenged with the-rapeutic compounds acid sphingomyelinase (ASMase) becomesactivated. The activated ASMase then translocates to membranesurfaces (Fig. 1) and hydrolyzes SM, which generates sphingo-sine and ceramide. This in situ breakdown of SM elevates cera-mide level, which rapidly displaces cholesterol from membrane/lipid-“chol-raft” and forms “cer-raft”. This newly formed “cer-raft” serves to sequester proteins of the FAS–DISC and relatedproteins, which immediately triggers “start” signals to death/apoptosis following endocytosis. The displaced cholesterol maymove to the other parts of the membrane enriched with phos-pholipid, and be continuously balanced by efflux of cellularcholesterol (See discussion and perspectives).

2. Lipid rafts/caveolae signaling and cancer

Epigenetic regulation of genes encoding raft components andits roles in cell transformation angiogenesis, immune escape, andmetastasis, and roles for rafts in other diseased states have beenreviewed earlier [1,5,8,13–15,24]. The role of cholesterol andrafts in non-genomic hormonal signaling in prostate cancer isintriguing and discussed recently [25,52]. This contribution is

Table 3Cav-1, CD44, E-cad and FAS interacting proteins in lipid rafts / caveolae mediatedsignaling

Proteins References, Link: http://www.ncbi.nlm.nih.gov/entrez/dispomin.cgi?id

Caveolin-1Integrin α-subunit [64]; 603963H-ras/Raf-1 [2,40,42,46]; 601619/164760Fyn tyrosine kinase [64]; 137025Ras-p42/44 MAPkinase cascade

[46,66]; see the caveolin link, 601047

SHC [64,104,108]; 600560EGF/EGFR [5,52,66,101,102,104]; 131530/131550GRB2 [64]; 108355Cyclin D-1 [67]; 168461Src-like kinases [1,5,8,49,65], 124095V-Src [8,46]; 190090PKA/PKC/PKC-α [48]; 601639, 609191/176982/176960

CD44Ezrin [1,4,76,77], 123900HA (hyaluronic acid) [76,77]; see 107269 and 600826Annexin-II [76,77]; 151740Rac-1 / Rac-2 [77]; 602048/602049

FASFASL [24,153,154,199–201,227,220]; 123900Ezrin [71,72,153,200] see 123900Caspase-8 and 10 [153,154,199,200,226,227] 151740


devoted to focus on the cellular biochemistry and signalingfunction of some protein components of membrane/lipid rafts incell transformation, tumor development and metastasis, whichwould help to best understanding our current concerns in raft-assisted signaling in regulation of cancer, and will help to selectstrategies for better management of neoplasm.

2.1. Raft and caveolin-1 signaling

Caveolin-1 (cav-1) protein has been identified as a markernecessary for lipid rafts/caveolae formation, stability andfunction, which is associated with multiple cellular processes innormal and pathophysiological state, including cancer progres-sion and hormone refractory diseases [1,3,5,8,13–17,27,53–55].In animal model studies targeted disruption of cav-1, andhomologous recombination created cav-1-null mice have shownthat natural presence of caveolae are absent from various celltypes resulting in severe physical limitations in cav-1-disruptedmice. Some of those include impaired nitric oxide and calciumsignaling in cardiovascular system, causing aberrations inendothelium-dependent relaxation, contractility, andmaintenanceof myogenic tone. In addition, cav-1 knockout mice lungdisplayed thickening of alveolar septa caused by uncontrolledendothelial cell proliferation and fibrosis [56,57]. Endothelialcav-1 and caveolae are necessary for both rapid and long-termmechanotransduction in intact blood vessels [53,58,61–63]. Lackof caveolae formation is associated with degradation andredistribution of cav-2, defects in the endocytosis of albumin (acaveolar ligand and a major plasma protein that transports andscavenges metabolites and endotoxic substances respectively),and a hyperproliferative phenotype in tissues and culturedembryonic fibroblasts from the cav-1-null mice [57,59]. Schubertet al. [60] has recently examined the role of "non-muscle"caveolins (cav-1 and cav-2) in skeletal muscle biology and foundthat skeletal muscle fibers from male cav-1(−/−) and cav-2(−/−)mice show striking abnormalities, such as tubular aggregates,mitochondrial proliferation/aggregation, and increased numbersof M-cadherin-positive satellite cells, which became morepronounced with ageing. Interestingly, they found that cav-2-deficient mice displayed normal expression levels of cav-1,whereas cav-1-null mice exhibited an almost complete deficiencyin cav-2. Hence, these skeletal muscle abnormalities seem to bedue to loss of cav-2. Histologic examination and echocardio-graphy identified a spectrum of characteristics of dilatedcardiomyopathy in the left ventricular chamber of the cav-1-deficient hearts, including an enlarged ventricular chamberdiameter, thin posterior wall, and decreased contractility [61].Cav-1 knockout (KO) mice were observed to be completelydevoid of caveolae. Lewis lung carcinoma cells implanted intocav-1 KO mice had increased tumor microvascular permeability,angiogenesis, and growth [62]. Cav-1 plays a crucial role in themechanisms that coordinate lipid metabolism with the prolif-erative response occurring in the liver after cellular injury camefrom an outstanding report that treatment of cav1-null mice withglucose, which is a predominant energy substrate when comparedto lipids, drastically increased survival and reestablishedprogression of the cell cycle [63].

2.2. Tumor suppression and metastasis promotion. The cav-1,CD44, E-cadherin paradoxes

Cav-1 functions as a membrane adaptor to link the integrinα-subunit to the Fyn tyrosine kinase. Upon integrin ligation,Fyn is activated and binds to SHC, via the SH3 domain of Fyn.SHC is subsequently phosphorylated at Y317 and recruits GRB2(Table 3). This sequence of events is necessary to couple inte-grins to the Ras–ERK (extracellular signal-regulated kinase)pathway and promote cell cycle progression [64]. Mutations incav-3 suggested that heritable differences in the interactionbetween caveolins and their partners may lead to other con-ditions, which mainly cause limb–girdle muscular dystrophy(Reviewed in ref. [8]).

Cell culture and biochemical findings evidenced that cav-1 isa tumor suppressor gene and a negative regulator of the v-Src,H-ras, Protein kinase A, PKC-isoforms, and Ras-p42/44 mito-gen activated protein (MAP) kinase cascade within caveolae([8,46] and references therein). Loss of heterozygosity analysisimplicates chromosome 7q31.1, (where cav-1 gene is localizedto a suspected tumor suppressor locus D7S522), in the patho-genesis of multiple types of human cancer, including breast,ovarian, prostate, and colorectal carcinoma, as well as uterinesarcomas and leiomyomas (reviewed in refs. [8,65], see also[66]). For example, cav-1 expression in mammary adenocarci-noma (MTLn3) cells inhibits epidermal growth factor (EGF)-stimulated lamellipod extension and cell migration, blocks theiranchorage-independent growth, and thus induces a non-motilephenotype by blocking the EGF-induced activation of the p42/44 MAP kinase cascade [66].

http://www.ncbi.nlm.nih.gov/entrez/dispomin.cgi?id

http://www.ncbi.nlm.nih.gov/entrez/dispomin.cgi?id


On the other hand, cav-1 can also function as tumor meta-stasis promoting molecule which is not likely to its cell growthinhibitory function ([46] and references therein, [67]). Forexample, higher expression of cav-1 induces filopodia forma-tion in lung adenocarcinoma with enhanced metastasis [46].Fig. 2 shows that concerted function of lipid rafts and cav-1 isnecessary for filopodia formation and an increase of metastaticpotential of lung adenocarcinoma cells. Cav-1 induces filopodiaformation with enhanced metastatic potential when lipid com-ponent of rafts virtually remains unaltered. Delipidation/cho-lesterol depletion disrupts cav-1 function and stopped filopodiaformation even when cav-1 protein was expressed in very highamount in cav-1+ve CL1-5 cells (Fig. 2B), C6 cells (Fig. 2C).Cav-1 expression is absent, or very weak in the low invasivelung cancer cell line CL1-0, which is in best agreement with theobservations that down regulated cav-1 expression facilitatescell transformation [1,46,47,62,66,67]. Down-regulation ofcav-1 is also associated with its 5′-proximal promoter CpG-island hypermethylation and tumor formation (reviewed in ref.[1]). Elevated cav-1 level is associated with lung, breast, pros-tate, and their lymph node metastases (see the link: http://www.ncbi.nlm.nih.gov/entrez/dispomin.cgi?id=176807), strengthen-ing the possibility that cav-1 may also act as an oncogene[1,46,68]. Thompson and colleagues [68–70] have demon-strated that cav-1 expression is significantly increased in pri-mary and metastatic human prostate cancer after androgenablation therapy, cav-1 is secreted by androgen-insensitiveprostate cancer cells, and that this secretion is regulated bysteroid hormones and the overall results established cav-1 as an

Fig. 2. Concerted function of lipid rafts and Caveolin-1 is necessary for filopodia formexpression of cav-1 induces filopodia formation with enhanced metastatic potential wdepletion disrupts cav-1 function and stopped filopodia formation even when cav-1 prand C7-Dox(−) cells (E) showed abundant filopodia formation. The cav-1−ve CL1-0 (tformation in cultures. However, the ability to form filopodia in CL1-5, C6 and C7-Dand H respectively). A rhodamine–phalloidin staining was used to highlight the presepermission from Ho et al. [46].

autocrine/paracrine factor that is associated with androgen-in-sensitive prostate cancer. It has also been suggested that cav-1might be a therapeutic target in the case of prostate cancer.Thus evidences are accumulating in favour of cav-1 as a directmediator of steroid action, signaling through PI3K–proteinkinase B (PKB, also designated as Akt) pathway, and metastasisof cancers [1,25,71]. Akt/PKB is a serine/threonine kinase thatis a critical regulator for cell survival and proliferation, especial-ly in human malignant cancers. Activated Akt phosphorylatespro-apoptotic proteins, thereby inactivating their activities. Aktactivation also up-regulates anti-apoptotic genes such as Bcl-XL

and FLICE-inhibitory protein (FLIP). Akt activation involvesphosphorylation of S473 and T308 by phosphoinositide-depen-dent kinases and integrin-linked kinase. Recent studies havesuggested that rafts have implications in Akt activation [71].

Puzzling question arises, why a tumor suppressor gene whoseinactivation is necessary for cell transformation and tumor in-duction can be re-expressed to facilitate tumor progression?Such a paradox is not restricted only to cav-1. It is apparent thatother molecules, including E-cadherin, CD44, granulocyte/macrophage-colony stimulating factor (GM-CSF), RAR-β2,and α- and β-catenins have also been reported to impart thevirtual opposite function in tumorigenesis ([46], reviewed inrefs. [1] and [72]). The promoters of the various cell surfaceadhesion marker genes, including cav-1, E-cad and CD44 areinactivated in association with promoter CpG-hypermethylationat the onset of tumor development and remains thereafter me-thylated in full-blown tumors but were found to be re-expressedin metastatic foci and lymph nodes. Since these genes are mostly

ation and increase of metastatic potential of Lung Adenocarcinoma cells. Highhen lipid component of raft virtually remains unaltered. Delipidation/cholesterolotein was expressed in very high amount. Cav-1+ve CL1-5 cells (B), C6 cells (C),he parental cells for C6 cells, A) and C7-Dox(+) cells (D) did not reveal filopodiaox(−) cells was abolished when cells were cultured in delipidated medium (F,G,nce of F-actin in filopodia and at the peripheral cytoplasmic area. Adapted with

http://www.ncbi.nlm.nih.gov/entrez/dispomin.cgi?id=176807

http://www.ncbi.nlm.nih.gov/entrez/dispomin.cgi?id=176807


inactivated by DNA methylation; their reactivations certainlyneed demethylation activity [1,47,72–74]. For instance, mRNAand protein expression of cav-1 is frequently lost in multiplecancers. At the cancer onset, cav-1 gene is repressed by DNAmethylation, while re-expression occurs just before metastasis[1,72–74]. CD44 is known to be involved in re-organization ofhighly dynamic structures of cytoskeleton when cells respond toextracellular stimuli by division and/or changes in shape oractivity [1,13,75–77]. CD44 is the major cell surface receptorfor hyaluronic acid (HA), co-localizes with annexin-II in the cellsurface lipid raft microdomains in their cytoplasmic face infibroblasts and blood cells. Both CD44 and annexin-II can bereleased from these lipid rafts by sequestration of cholesterolwith altered cytoskeleton, prevention of Rac1, and prevention ofRac1-induced lamllipodia outgrowth, which was activated byHA binding to CD44 [76,77]. mRNA and protein expression ofCD44 is frequently lost in multiple cancers at the early stage ofcell transformation and tumor progression, which also is as-sociated with DNA hypermethylation [1,72,78–81]. Again, re-expression of CD44 gene was found necessary for metastaticdiffusion of many tumors [1,72,81–85]. The transmembraneglycoprotein E-cad, a calcium-dependent cell–cell adhesionmolecule, is known to play a key role in the maintenance of tissueintegrity by forming complexes with catenins (α, β, and γ). E-cadis eventually tagged to actin cytoskeleton through catenins.Because loss of E-cad expression results in disruption of cellularclusters, it has been postulated that E-cad functions as tumorsuppressor gene. mRNA and protein expression of E-cad isfrequently lost onDNAmethylation inmultiple cancers at the earlystage of tumor progression [1,72,81,86–94]. Also in this case, re-expression of E-cad had been shown to be clinically significant atthe metastatic foci of many cancers [1,72,74,85,91,92,95]. Cavalliet al. [95] have studied the epigenetic features in breast sentinellymph nodes (SLN) compared with their corresponding matchprimary tumors for CpG-island hypermethylation of a few genes,including THBS2, E-Cad, and RAR-β2 in six paired primarybreast tumors and their matched SLN. They noticed that, overall,71% (30/42) of the methylation measurements were identicalbetween the primary tumors and the SLN. Dissection of suchalterations may lead to identification of initial events associatedwith the metastatic dissemination process.

3. Raft and epidermal growth factor receptor (EGFR)signaling

Cohen (1962) first described a growth factor that has aprofound effect on the differentiation of specific cells in vivo,and which is a potent mitogenic factor for variety of culturedcells of both ectodermal and mesodermal origin that is knowntoday as epidermal growth factor (EGF) or Urogastrone (URG).URG/EGF (OMIM 131530) is also a potent inhibitor of gastricacid secretion and promotes epithelial cell proliferation. MatureEGF is a single-chain polypeptide consisting of 53 amino acidsand having a molecular mass of about 6 kDa only [96,97].

The EGF receptor (EGFR) is a 170 kDa transmembrane lipidraft glycoprotein comprising a 1186 amino acid polypeptidechain and is composed of three domains: an extracellular ligand-

binding domain, a single transmembrane lipophilic region, andan intracellular domain that exhibits intrinsic tyrosine kinaseactivity [98–104]. The extracellular ligand-binding region onthe cell membrane is connected to the intracellular machinerythat possess the tyrosine kinase activity via a single hydro-phobic membrane domain [98,99]. Helin et al. have demon-strated that the biological activity of the human EGFR ispositively regulated by its C-terminal tyrosines [100]. Endo-genous ligands to EGFR include TGF-α, heparin-binding EGF,amphiregulin and betacellulin [105]. It transmits signals uponactivation by complex formation with the cognate ligand, EGF,or in some instances activated by cross-talk with the otherligands of receptor tyrosine kinases (RTKs) family, namelyTGF-α [97–99]. On binding of the ligand to EGFR, the ligand–receptor complex undergoes dimerization and internalization[104,106,107]. The EGFR family plays an essential role innormal organ development by mediating morphogenesis anddifferentiation, and plays a crucial role in growth, differentia-tion, and motility of normal as well as cancer cells.

Yamabhai and Anderson [102] have identified that thesecond cysteine-rich region of EGFR contains targeting infor-mation for caveolae/rafts. Puri et al. [104] have shown thatseveral endocytic proteins are efficiently recruited to morpho-logically distinct plasma membrane lipid rafts, upon activationof EGFR. Analysis of detergent-resistant membrane fractionsrevealed that the EGF-dependent association of endocyticEGFR proteins with rafts is as efficient as that of signalingeffector molecules, such as GRB2 or SHC [104,108]. Spe-cialized membrane microdomains have the ability to assembleboth the molecular machineries necessary for intracellularpropagation of EGFR effector signals and for receptor inter-nalization. In the case EGFR the localization of the two pro-cesses is well characterized ([104] and references therein).Signaling occurs within specialized membrane microdomains,lipid rafts [5,13], whereas endocytosis occurs mostly throughthe clathrin-coated pits [104,106,109]. The signal transductionpathways can lead to cell proliferation and tumor growth, as wellas progression of invasion and metastasis [98,105,107,110].Fig. 3 (right half) shows a schematic view for how EGFRsignaling cascade operates and where “chol-rafts/caveolae” areinvolved. Overexpression of EGFR produces a neoplasticphenotype in tumor cells, which is associated with higher ratesof progression from superficial to invasive forms of variouscancers [1,66,105,107,111–114]. Nagane et al. [115] havereported that a common mutant EGFR confers enhanced tumor-igenicity on human glioblastoma cells by increasing prolifera-tion and reducing apoptosis. Thomas et al. [116] have reportedthat cross-talk between G protein-coupled receptor (GPCR) andEGFR signaling pathways contributes to growth and invasion ofhead and neck squamous cell carcinoma (HNSCC). Combinedblockade of both EGFR and GPCRsmay be a rational strategy totreat cancers, including HNSCC that shows cross-talk betweenGPCR and EGFR signaling pathways [116]. Scartozzi et al.[117] have analyzed the expression of activated (phosphory-lated) Akt and MAPK in 98 cases of paired primary colorectaltumours and metastases with the aim to define better the EGFR-related molecular profile of colorectal cancer (CCR) as a tool for


treatment selection. EGFR downstream signaling pathway canbe hyper activated even in the absence of EGFR expression in aconsiderable proportion of patients, which indicates cross-talksignaling. Curto et al [118] have shown that upon cell–cellcontact neurofibromatosis type 2 (NF2) tumor suppressors,Merlin, coordinates the processes of adherens junction stabiliza-tion and negative regulation of EGFR signaling by restrainingthe EGFR into a membrane compartment from which it canneither signal nor be internalized. The (n-3) fatty acids (FA),eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)decrease proliferation and induce apoptosis in MDA-MB-231human breast cancer cells. Schley et al. [119] have examined theeffects of EPA and DHA on the lipid composition of lipid rafts aswell as raft localization of EGFR, and phosphorylation of EGFR.EPA and DHA treatment decreased lipid raft sphingomyelin,cholesterol, and diacylglycerol content; and in the absence oflinoleic acid, both EPA and DHA increased ceramide levels inraft. Furthermore, there was a marked decrease in EGFR levels in

Fig. 3. Raft signaling to survival and death. Right panel: Lipid rafts/Caveolae can brEGFR (a “chol-raft” protein) initially trigger recruitment of the GRB2/Sos complexErk, which translocates to the nucleus to phosphorylate Elk-1 and other transcriptionprocess, and rate of which reaction depends on the types of signals cells receive. Howinternalization, which may be the rate limiting step. At the same time transformation oInternalization of FAS–DISC in association with “cer-raft” leads to death. See the te

lipid rafts, accompanied by increases in the phosphorylation ofboth EGFR and p38 MAPK, in EPA+DHA-treated cells. Assustained activation of the EGFR and p38 MAPK had beenassociated with apoptosis in human breast cancer cells, theirresults indicate that (n-3)-FA modify the lipid composition ofmembrane rafts and alter EGFR signaling in a way that decreasesthe growth of breast tumors. The result is intriguing and showsantithetic effects of EGFR signaling on cell regulation. Oh et al.[120] have investigated whether membrane cholesterol couldregulate apoptosis, and elucidated a mechanism by which apop-tosis is induced in prostate cancer cells. When LNCaP cells wereexposed to 2-hydroxypropyl-beta-cyclodextrin (HPCD), cellviability was inhibited by HPCD dose dependently, and restoredby replenishment of cholesterol. Caspase-3 and PARP cleavageassays suggested that cells were dying on application of HPCD byapoptotic cell death through down-regulation of Bcl-XL, andinhibition of both EGFR/Akt and EGFR/ERK signal transductionpathways, which indicated the lowering of the pool of “chol-raft”.

eak the MAP kinase pathway at multiple points. Growth factor receptors, e. g.,to the plasma membrane leading to sequential activation of Ras, Raf, MEK andfactors. See the text for details. Left panel: FAS-ligation is comparatively a slowever, once FAS–FASL complex is formed, FAS interacts with Ezrin very fast forf “chol-raft” to “cer-raft” is over, for which involvement of ASMase is necessary.xt for details and compare with Fig. 4.


4. Nuclear factor kappa B signaling

The oxidative stress sensitive transcription factor playingcritical roles in the regulation of a variety of genes, important inmultiple cellular responses, is the nuclear factor-kB (NFκB).NFκB remains inactive in the cytoplasm sequestered through itsinteraction with IkB and became activated in inflammation andcancer. IkB kinase phosphorylates IkB and subsequent ubi-quitination cause degradation of the latter. Eventual release ofNFκB is followed by its translocation to the nucleus. The NFκBinducing kinase controls activation of IkB kinase, followed by across-talk between activation of the extracellular signal-regulatedkinase (ERK)/MAP kinase pathway, and the NFκB-inducingkinase/IkB kinase/NFκBpathway.NFκB is reported to be directlyassociated with lipid rafts by Misra et al. [121]. They haveobserved that, in T-cells, following TCR ligation, a small portionof total cellular caspase-8 and c-FLIPL rapidly migrate to lipidrafts where they associate in an active caspase complex. Acti-vation of caspase-8 in lipid rafts is followed by rapid cleavage ofc-FLIPL at a known caspase-8 cleavage site. The active caspase/c-FLIP complex forms in the absence of FAS and recruits theNFκB signaling molecules RIP1, TRAF2, and TRAF6, as well asupstream NFκB regulators PKC, CARMA1, Bcl-10, andMALT1, which connect to the TCR. Inhibition of caspaseactivity, furthermore, blocks NFκB activation. These findingsdefine a link among TCR, caspases, and the NFκB pathway thatoccurs in a sequestered lipid raft environment in T-cells [121].Disruption of lipid rafts might deregulate the above kinase-axis,sinceMAPkinase function depends largely on its associationwithlipid rafts/caveolae [8,46,57,58] (See Fig. 3). Because based onrecent studies, NFκB is considered as a target for the managementof cancer, modulation of this pathway by targeting lipid raftscould also contribute to its preventive potential (See Discussionand perspectives for further information).

5. Rafts in MAP kinase, Ras, and activator protein 1 (AP1)signaling

MAP kinases have been shown to play important roles inmany cellular physiologic processes, including proliferation,differentiation, and survival or death. In mammalian cells thereare the three major types of MAP kinases: (i) c-Jun NH2-terminal kinases (JNK), (ii) p38 MAP kinases, and (iii) ERK.Activation of ERK1 and ERK2 (ERK 1/2) in this pathwaymodulate a wide variety of cellular activities via the regulationof several transcription factors. The ability of the ERK/MAPKpathway to promote cell growth by activation of cyclin D iscounterbalanced by the concomitant production of the cyclin-dependent kinase inhibitor p21WAF1. Moderate activation of thepathway leads to cell proliferation, while hyperactivation resultsin p21WAF1-mediated growth arrest. In addition, induction ofthe cell cycle inhibitory INK4 proteins, including p16INK4A, ismediated by the Ras/Raf/MEK/ERK pathway (Fig. 3, righthalf). Several studies have implicated that overexpression andactivation of ERK/MAPK plays an important role in coloncancer progression, which may be a useful molecular target forcolon cancer therapy.

The major pathways that lie down stream of the membrane-associated receptor tyrosine kinases (RTK) is activation of Raf-1by lipid raft associated Ras [5,122], which follows phosphoryla-tion mediated activation of MAPK/ERK kinase 1/2 (MEK1/2).Activated MEK1/2 then phosphorylates ERK1/2. The JNK1/2/3and p38α/β/γ pathways are parallel MAP kinase cascades inmammalian cells [46,121–125]. Once activated, MAP kinases(ERK, JNKand p38) activate nuclear transcription factor E-26-likeprotein (ELK) and c-Jun [123,126]. Phosphoinositide 3-kinase(PI3K) is activated by RTKs and it synthesizes the secondmessenger, phosphatidyl inositol-3,4,5-triphosphate, which isnecessary for phosphorylation of Akt (protein kinase B). Aktdirectly phophorylates and inactivates the pro-apoptotic proteinBad, and enhances the antiapoptotic function ofBcl-XL. In LNCaPhuman prostate cancer cell line, it was shown that lipid raftsmediate Akt-regulated survival [1,123,127].

Raf-1 also is a component of lipid rafts, and because thederegulated overexpression of MAP kinase pathway is fre-quently seen in a variety of human cancers, modulation of MAPkinases by disruption of lipid rafts along with the use of somenatural compound, therapeutic agents and other inhibitors,including MEK1/2 inhibitor PD98059 (2-(2′-amino-3′-methox-yphenyl) oxanaphthalen-4-one), and the PKC-δ inhibitorrottlerin may provide novel strategies for the prevention andtreatment of cancer. AP-1 transcription factor is a heterodimerof leucine zipper super family proteins, specially, heterodimerof the c-Jun and c-Fos proteins. High AP-1 activity has alsobeen shown to be involved in the tumor induction and progres-sion of various types of cancers, including colon, lung, breast,skin and prostate cancers ([123] and references therein).

6. Raft and insulin like growth factor mediated signaling

Insulin like growth factor (IGF) family of ligands, associatedproteins and its receptors is a significant essential growth factorsystem involved in the maintenance of cellular functions.Masticket al [128] have shown that insulin stimulates the tyrosinephosphorylation of caveolin. The coupling of free IGFs to IGF-1results in intracellular receptor autophosphorylation and phos-phorylation of precise downstream targets, which leads to crossactivation of several signaling pathways, including PI3K/Aktpathway and the Ras/MAP kinase pathway, both of which havecomponents docked in lipid rafts (Table 2). This leads to acti-vation of cell proliferation by inducing specific genes and DNAreplication. Therefore targeting the IGF-1 signaling pathwaythrough lipid rafts might be an effective strategy for preventionand cure of few cancers (reviewed in refs. [5,123], see also [128]).

7. Extracellular matrix and raft signaling

The ability of cells to respond appropriately to environ-mental cues is critical to maintaining cellular, tissue and or-ganism homeostasis. One such environmental cue is derivedfrom cellular adhesion to the extracellular matrix (ECM). Theloss of adhesion-dependent cellular regulation can lead to in-creased cellular proliferation, decreased cell death, changes incellular differentiation status, and altered cellular migratory


capacity; all of which are critical components of cellular car-cinogenesis andmetastasis of the disease [28–32,125,129–135].

8. Rafts in MMPs and uPAR signaling

The invasive nature of tumor cells vexed and bedeviled thecurrent treatment pathways, as the remaining tumor cells in-evitably invade the surrounding normal tissues, which leads totumor recurrence. Such local invasion remains to be an im-portant cause of mortality and underscores the need to under-stand in depth the mechanisms of invasion. Several proteasesinfluence the malignant characteristics of various carcinomas,gliomas and sarcomas — their inhibition could prove to be auseful therapeutic strategy [1,129–135].

Extracellular matrix is a key component of the tissuedestroyed by tumor-cell invasion. This is a dynamic environ-ment that has a pivotal role in regulation of cellular functionsduring normal and pathologic remodeling processes, such asembryonic development, tissue repair, inflammation, and tumorinvasion and metastasis. Protease profiling studies have in-dicated that expression of the serine protease urokinase-typeplasminogen activator (uPA) and uPA receptor (uPAR) anauthentic lipid–raft protein [1,36], and expression of the cys-teine protease cathepsin-B, and of the matrix metalloproteinasesMMP2 andMMP9 is increased in high-grade cancers, includingprostate, breast, and astrocytomas compared with respectivelow-grade prostate, breast, and astrocytomas, and the normalprostate, breast, and brain [129–134]. Strategies to prevent theexpression of uPA and uPAR at the molecular level have led tosignificant reduction/inhibition of tumor invasion and growth.Down-regulation of MMP2 and MMP9 expression throughapproaches such as MMP inhibitors or antisense vectors resultsin less tumor cell invasion and the inhibition of tumor growthand angiogenesis. Aberrant, persistent inclusion into lipid raftslimits the tumorigenic function of MMP1 in malignant cells(reviewed recently, [1]). Studies with cathepsin-B using itsnatural inhibitor, cystatin-C and antisense vectors have shownsignificantly to reduce tumor formation and invasiveness. Theseproteases may have interacted with each other and directly orindirectly can facilitate the expression of other proteases, assuch; the inactivation of one molecule seems to cause reducedexpression of the other molecule and/or the entire pathways[51–54,121–123,129–136]. The progression of human tumorsinvolves the MMP family. Two particular members of thefamily, MMP2 and MMP9, seem to have an important role ininvasion and metastasis of varied tumors [30,129–134,137–141]. DNA methylation has also been shown to affect MMPsexpression [140].

uPA is a trypsin-like protease that converts the zymogenplasminogen into active plasmin. It has the ability to preventapoptosis, stimulate angiogenesis, mitogenesis, cell migration,and to modulate cell adhesion. Inhibition of urokinase can de-crease tumor size or even complete remission of cancer in mice.The known urokinase inhibitors are highly toxic and need highconcentration for effective inhibition. It has been reported bymolecular modeling that tea polyphenol EGCGblocks urokinaseactivity by interacting with H57 and S195 extending toward R35

from a positively charged loop of urokinase thus prevents theformation of the catalytic triad of urokinase [123,129,142]. Thusdestabilization of uPAR function by disturbing its raft assemblymay help blocking of uPA activity, one of the most frequentlyoverexpressed enzymes in human cancers.

9. Rafts in integrin and focal adhesion kinase mediatedsignaling

The focal adhesion kinase (FAK) family kinases (whichinclude FAK and pyk2) regulate cell adhesion, migration, andproliferation in a variety of cell types ([136], reviewed in refs.[143,144]). Adhesion of cells to the ECM is mediated byheterodimeric transmembrane integrin receptors located withinsites of close apposition to the underlying matrix called focaladhesions. Integrin engagement and clustering stimulates FAKphosphorylation on Y397, creating a high affinity binding site forSrc and Src family kinases, the premier lipid raft associatedfamily of kinases [1,5,13,135,136,143–146]. The FAK/Srccomplex phosphorylates many components of the focal ad-hesion, resulting in changes of adhesion dynamics and the ini-tiation of signal cascades. In addition to FAK catalytic activity,FAK also functions as a scaffold to organize structural andsignaling proteins within focal adhesions.

The importance of FAK as a regulator of normal cellularfunction is underscored by the number of cancers reported tohave alterations in FAK expression and/or activity, includingprostate, colon, breast, cervical, ovarian, head and neck, colon,liver, stomach, sarcoma, glioblastoma and melanoma, and inaddition alterations in FAK expression and/or activity have beenassociated with tumorigenesis and increased metastatic poten-tial [136,143–146]. Currently, it is unclear how the catalyticactivity and/or scaffolding function of FAK contribute to tu-mor progression. To date studies of FAK function relied onexpression of dominant interfering mutants or elimination ofFAK expression by gene knockout, repression by antisenseoligonucleotide or siRNA. A recent report on the biochemicaland cellular characterization of a novel small molecule inhibitor,PF573,228 that targets FAK catalytic activity shows that theinhibitor interacts with FAK (recombinant or endogenous) inthe ATP-binding pocket and bloks its phosphorylation at Y397

position in a variety of normal and cancer cell lines [136].Babuke and Tikkanen [147] recently have reviewed the role

of reggie and flotillin (flot) in various cellular processes such asinsulin signaling, T-cell activation, membrane trafficking, pha-gocytosis, and EGFR signaling. Reggie-1/flot-2 and reggie-2/flot-1 are ubiquitously expressed and their molecular functionsdepend on post-translational modifications such as phosphor-ylation and lipid modifications. Hazarika et al. [148] havedemonstrate that overexpression of flot-2, alters the phenotypeof SB2 melanoma cells and is associated with up-regulation ofthe GPCR for thrombin, PAR-1. Although flot-2 is also ex-pressed in normal melanocytes, these data suggest that the levelsof flot-2 increase with melanoma progression in cell lines and inmelanocytic lesions. PAR-1 is upstream to the B-Raf/MAPK/ERK pathway and implicated in melanoma progression. Onemight expect that flot-2 may influence other GPCR, so that


modulation of PAR-1 by flot-2 may have significance for signaltransduction beyond this specific tumor model [147–149].

10. Raft signaling and apoptosis

Programmed cell death (apoptosis) is a highly ordered pro-tective mechanism through which unwanted, fatigued or dam-aged cells are eliminated from the system, which is essential fornormal development, immunological competence, and home-ostasis. Moreover, apoptotic cell death, which is preceded bythe activation of effector proteases, known as caspases, whichresults in the cleavage of varied endogenous proteins involvedin structure maintenance and signal transduction in an orga-nism. Also, apoptosis serves as a protective mechanism againstneoplastic developments by eliminating genetically damagedcells or excess cells that have improperly been induced todivide. It is characterized by marked changes in cellular mor-phology, including chromatin condensation, membrane bleb-bing, nuclear breakdown and the appearance of membraneassociated apoptotic bodies, internucleosomal DNA fragmenta-tion, cleavage of poly(ADP-ribose) polymerase (PARP), aswell as by release of hypoacetylated and trymethylated histoneH4 [122,132,150–156].

For many cancers, including lung, cholangiocarcinoma, andcancer of endothelial cells when in advanced malignant stage,treatment/management options become restricted to radiationtherapy and/or chemotherapy. Current data on the molecularmechanisms of radiation/chemo-induced tumor cell killing sug-gest that tumor cell destruction follow apoptotic pathway throughtransformation of “chol-raft” to “cer-raft”, and “cer-raft”-mediated FAS–DISC internalization.

11. FAS and raft mediated FAS signaling

cDNA encoding the Human FAS antigen (TNFRSF6/FAS/APT1/APO1/CD95, OMIM 134637) consist of a 16-amino acidsignal sequence followed by amature protein of 319 amino acidswith a single transmembrane domain and a molecular mass ofapproximately 36 kD [157]. The protein contains three domains;a FAS death domain, a FAS ligand (FASL) binding domain, andthe transmembrane domain. The FAS antigen shows structuralhomology with a number of cell surface receptors, includingtumor necrosis factor (TNF) receptors and the low-affinity nervegrowth factor receptor (NGFR). Northern blot analysis detected2.7 kb FAS mRNAs in thymus, liver, ovary, and heart. Func-tional expression studies in mouse cells showed that the FASantigen induce antibody (FASL)-triggered apoptosis. Canaleand Smith [158], reported defect in the FAS gene in a patientwith autoimmune lymphoproliferative (lpr) syndrome (ALPS;OMIM, 601859), which was due to mutation. Drappa et al. [159]confirmed that in the patients affected son, who had aheterozygous 972G-to-T transversion within the death domainof the FAS gene, resulting in a D244Y substitution. Subse-quently, it had been shown that the FAS receptor can rapidly beexpressed on T-cells following activation of T-cell hybridomas,and that the interaction between FAS and FASL-induced celldeath occurs in a cell-autonomous manner consistent with

apoptotic features [160,161]. Mannick et al. [162] have de-monstrated that FAS activates caspase-3 by inducing thecleavage of the caspase zymogen to its active subunits and bystimulating the denitrosylation of its active site thiol. MYC-induced apoptosis requires interaction between FAS and FASLon the cell surface [163]. The findings of Hueber et al. linked thetwo apoptotic pathways, establishing the dependence of MYCon FAS signaling for its killing activity. The signaling pathwayleading to apoptosis by FAS cross-linking with FASL results inthe formation of a death-inducing signaling complex (DISC)composed of FAS, the signal adaptor protein FAS-Associatedvia Death Domain (FADD), and procaspase-8 and 10, and thecaspase-8/10 regulator c-FLIP [164,165]. This associationgenerates Casp8/10, activating a cascade of caspases. Lepple-Wienhues et al. [166] have demonstrated that in addition to therole of FAS in inducing cell death, stimulation of FAS inhibitsthe influx of calcium normally induced by activation of the T-cellantigen receptor, in part, by not affecting the release of calciumfrom intracellular stores. This block in calcium entry can bemimicked by stimulating T-cells with acid sphingomyelinase(ASMase) metabolites of the plasma membrane lipid sphingo-myelin, such as ceramide and sphingosine [166]. Grassme et al.[167] have showed that Pseudomonas aeruginosa infectioninduced apoptosis of lung epithelial cells were by activation ofthe endogenous FAS/FASL system. Deficiency of FAS or FASLon epithelial cells prevented apoptosis of lung epithelial cells invivo as well as in vitro.

Many studies have indicated that perhaps all tumor cellsexpress FAS, but with mutations. However, Arscott et al. [168]have demonstrated that the FAS antigen is expressed andfunctional in papillary thyroid cancer cells. Lee et al. [169] haveanalyzed the entire FAS coding region by microdissectiontechniques of biopsy samples from 21 burn scar-related squa-mous cell carcinomas (BSRSCC) with somatic point mutationsin all of the splice sites from 3 patients. The FAS mutations werelocated within the death domain, ligand-binding domain, andtransmembrane domain. No mutations were detected in 50 casesof conventional SCC [169]. Loss of heterozygosity (LOH) of theother FAS allele was demonstrated in tumors carrying the N239DandC162Rmutations, and expression of FASwas confirmed in alltumors with FAS mutations. BSRSCC is usually more aggressivethan conventional SCC, and Lee et al. have suggested that somaticmutations in FAS may contribute to the development and/orprogression of BSRSCC and identified following in the FAS gene:a 957A-to-G transition resulting in an N239D substitution in theFAS death domain; a 547A-to-G transition resulting in an N102Ssubstitution in the FAS ligand-binding domain; a 726T-to-Ctransition resulting in a C162R substitution in the FAStransmembrane domain. Zhang et al. in 2005 have genotyped1,000 Han Chinese lung cancer (211980) patients and 1,270controls for 2 functional polymorphisms in the promoter regionsof the FAS and FASL genes, -1377G-to-A (134637.0021) and-844T-to-C (134638.0002), respectively. Compared to noncarriers, there was an increased risk of developing lung cancerfor carriers of either the FAS -1377AA or the FASL -844CCgenotype; carriers of both homozygous genotypes had amore than4-fold increased risk [170]. Their results support the hypothesis


[171], that the inactivation of FAS- and FASL-triggered apoptosispathway plays an important role in human carcinogenesis.

In animal model studies of lpr mice showed that FAS is thegene for the mouse lpr, and is identical to human patientsdisplaying a phenotype similar to that of lpr mice [172,173].The murine phenotype autosomal recessive lpr was character-ized by lymphadenopathy, hypergammaglobulinemia, multipleautoantibodies, and the accumulation of large numbers ofnonmalignant CD4-, and CD8-T-cells. Affected mice usuallydeveloped a systemic lupus erythematosus-like autoimmunedisease, and a defect in the negative selection of self-reactive Tlymphocytes in the thymus. Autoimmune disease in mice maybe due to integration of endogenous retrovirus in the FAS gene[174]. The importance of FAS in the pathogenesis of diabeteswas evaluated by generating non-obese diabetic mice thatdevelop spontaneous autoimmune diabetes, with beta cell-spe-cific expression of a dominant-negative point mutation in theFAS death domain [175]. In vivo silencing effect of siRNAduplexes targeting the FAS gene to protect mice from liverfailure and fibrosis in two mouse models of autoimmunehepatitis, has indicated that FAS siRNA specifically reducedFAS mRNA and protein levels in mouse hepatocytes whichwere resistant to apoptosis [176]. It is observed that FAS-defi-cient lpr −/− mice had less severe collagen-induced arthritis,possibly by blocking the IL1R1 or TLR4 (603030) pathway thatis generally activated by FAS–FASL interaction [177]. Landauet al. have pointed that FAS-deficient lpr mice developed aParkinson's disease phenotype [178].

Fisher et al. [179] have identified a heterozygousmutation in theFAS gene in 5 unrelated children (134637.0001–134637.0005),with a rare ALPS. The disorder was characterized by massivenonmalignant lymphadenopathy, autoimmune phenomena, andexpanded populations of TCR–CD3(+)CD4(−)CD8(−) lympho-cytes, and each child had defective FAS-mediated T lymphocyteapoptosis in vitro. One of the patients studied by Fisher et al. wasincluded in the report by Sneller et al., who were delineating thisdisorder and pointed out its resemblance to autosomal recessive lpr/gld (generalized lymphoproliferative disorder) disease in themouse. The lpr and gld mice bear mutated genes for FAS andFASL, respectively [180].Many other investigators have identifiedframe shift mutations due to base deletions or insertions in variousexons coding regions, LOH as well as single nucleotide poly-morphism of the FAS gene from ALPS (type IA, IB, II and III)patients having multiple phenotypic features of ALPS, includingelevated numbers of double-negative T-cells, hypergammaglobu-linemia, and FAS mutations were found in a fraction of CD4+ andCD8+ T-cells, monocytes, and CD34+ hematopoietic precursors,and B-cell lymphoma ([169,181–188], for an exciting review see[189]). Siegel et al. have found dominant interference of FASmutations in the first cysteine-rich domain that results ligand-independent molecular interaction of wild type and mutant FASreceptors through a specific region of the extracellular domain,rather than depending upon FASL-induced FAS oligomerization,in a preassociated receptor complex which normally permits FASsignaling [188–190].

Epigenetic DNA-methylation studies on FAS expression andsensitivity to its ligation on murine CD8 cells specific for the

CW3 antigen expressed by transfected P815 cells implicatedthat loss of FAS expression by antigen-specific cytotoxic T-cellsmay be due to DNAmethylation [191]. In an attempt to evaluatethe potential role of FAS gene as a model of gene therapyShimizu et al. [192] have evaluated genetic and epigeneticevents leading to alternation of the introduced FAS gene. Theynoticed solid tumors formed by FAS cDNA-transfected hepa-toma cells, F6b, were almost completely cured by a singletreatment of anti-FAS monoclonal antibody (mAb) but recurredin gld/gld–lpr/lpr mice after initial complete response. Recurredtumors were resistant to repeated mAb treatment. The FAS-resistant tumor contained two types of cells without FASprotein, and with decreased FAS protein. Cells having no FASwere due to the deletion of FAS cDNA. However, FAS-decreased cells retained FAS cDNA, which was highlymethylated. Petak et al. [193] have demonstrated that DNAhypermethylation is one mechanism that contributes to the down-regulation of FAS expression and subsequent loss of sensitivity toFAS-induced apoptosis in colon carcinoma cells. Impairedfunction or repression of FASL may inactivate FAS stimulationand facilitate tumor growth. Castellano et al. [194] have shownthat active transcription of the human FASL/TNFSF6 promoterregion in T lymphocyteswas involved in chromatin remodeling inassociation with DNA methylation. The resistance of small celllung cancer (SCLC) cells by FASL and TRAIL induced apoptosishas been explained by an absence of FAS and TRAIL-R1 mRNAexpression and a deficiency of surface TRAIL-R2 protein [195].In addition, caspase-8 expression was absent, whereas FADD,FLIP and caspases-3, -7, -9 and -10 were detectable. Loss ofmRNA and protein expression was well correlated with DNAmethylation of the respective genes encoding FAS, Casp-8 andTRAIL-R. Peli et al. [196] have nicely demonstrated that theoncogenic potential of H-rasmay reside on its capacity not only toparticipate in the mitogen activated signaling cascade to promotecellular proliferation (Fig. 3, right panel), but also to simul-taneously inhibit FAS-triggered apoptosis. They reported thatoncogenic Ras (H-Ras) downregulated FAS mRNA and proteinexpression in association with DNA methylation, and renderedcells of fibroblastic and epitheloid origin resistant to FASL-induced apoptosis. They suggested that Ras signals via the PI3-kinase pathway to downregulate FAS, suggesting that the knownanti-apoptotic effect of the downstream PKB/Akt kinase may bemediated, at least in part, by the repression of FAS expression.Many neuroblastomas have hypermethylation and down-regula-tion of CASP8, leading to resistance to TRAIL. van Noesel et al.[197] have analyzed methylation of multiple genes in 22neuroblastoma cell lines. In 40% neuroblastoma cells FLIPgene adjacent to CASP8 map at chromosome 2q33 wasmethylated. Down-regulation of FLIP strongly correspondswith down-regulation of Casp-8, and this was also found forDCR1 and DCR2. The FLIP protein is a negative regulator ofCasp-8, and its methylation patterns showed a moderate cor-relation. Co-methylation patterns were observed for the TRAILreceptor pairs DCR1 and DCR2, and between DR4 and DR5. Allfour receptors co-localize in chromosome band 8p21. Thehypermethylation status of the FAS promoter in prostatic andbladder carcinomas and respective cell lines appears to play a role


in down-regulation of FAS expression, which might be one of thecauses of tumor formation in prostate and bladder [198].

The ability of FAS signaling to mediate death signals and toplay a critical role in development pathways may hinge on itsability on downstream caspase activation. Two reasonable can-didates for mediating the regulation of signaling consequencesmay be c-FLIP, and ASMase. C-FLIP at high concentrations caninhibit FAS-mediated apoptosis ([199] and references therein).Another possible switch between pro-apoptotic and non-apoptoticFAS signaling may be the post-translational modifications thatregulate the ability of FAS to become internalized after itsactivation by FASL. Two acceptor sites for post-translationalmodifications in the intracellular domain of FAS may have im-portance in apoptotic activities of FAS. C199 regulates twoprerequisite steps for FAS internalization, localization of thereceptor to lipid rafts and FAS aggregation [200], and Y291 isimportant for internalization of FAS [201]. Simultaneously,ASMase translocation to lipid raft may cause ceramide ac-cumulation by catalytic hydrolysis of SM to ceramide andsphingosine. This locally produced ceramide remodels raft to formlarger domains of “cer-raft” and promotes rapid aggregation ofFAS as well as FAS–FASL complex formation [20,22–24,166].Internalization of FAS into lipid raft or an endosomal compart-ment may determine which signaling pathways are involved.When internalization of FAS is blocked, the receptor cannotinduce apoptosis and instead remains fully engaged, may be, inactivating non-apoptotic pathways [201]. FAS, therefore, seems tobe similar to other internalizing receptors such as the EGFR.Internalization of EGF target receptors, EGFR, and EGFRdownstream receptor sequestration in the lipid rafts or endocyticcompartmentmay thus contribute to both the intensity of signalingand assembly of signaling complexes [104,202].

12. Rafts and ezrin signaling

Ezrin (OMIM 123900) is a component of the microvilli ofintestinal epithelial cells that serves as a major cytoplasmicsubstrate for certain protein–tyrosine kinases. It is the same ascytovillin (CVL), which is a microvillar cytoplasmic peripheralmembrane protein that is expressed strongly in placental syn-cytiotrophoblasts and in certain human tumors. cDNA cloning,sequencing, and deduction of protein sequence indicated thathuman ezrin is a highly charged protein with an overall pI of 6.1and a calculated molecular mass of 69 kDa, which is close toserum albumin [59,203,204]. A 3.2-kb ezrin mRNA is known toexpress the protein with a relative highest level in intestine,kidney, and lung cells. The cDNA clone hybridized to DNAsfrom widely divergent organisms, indicating that the sequenceis highly conserved throughout evolution. Within its N-terminaldomain, ezrin showed a high degree of similarity of amino acidsequence to the erythrocyte cytoskeletal protein band 4.1(OMIM 130500). Saotome et al. [205] have found that con-ditional mutation in the ezrin gene gave birth of homozygousmutant mice at sub-Mendelian ratios (about 12%), but despitetheir normal appearance at birth they failed to thrive and did notsurvive past weaning. The luminal surface of ezrin −/− intestinewas covered with cauliflower-shaped villi that appeared to be

aggregates of multiple individual villi. They observed that themorphologic complexity of the ezrin −/− villi increased withpostnatal age, leading to the formation of complex amalgamatedstructures, but the establishment and maintenance of epithelialpolarity was not affected. Hence, it was concluded that ezrin isnot absolutely required for the formation of brush bordermicrovilli, but it performs a critical function in organizing theapical domain of the intestinal epithelial cell and its associatedapical junctions [205]. Ezrin, radixin (RDX; OMIM 179410),and moesin (MSN; OMIM 309845), the so-called ERM pro-teins, act as linkers between the plasma membrane and the actincytoskeleton. They are involved in a variety of cellular func-tions, such as cell adhesion, migration, and the organization ofcell surface structures. They are highly homologous, both inprotein structure and in functional activity, with merlin/schwan-nomin, the NF2 tumor suppressor protein [206]. Ezrin is pre-dominantly a raft protein [4,36]. Gupta et al. [4] have recentlyanalyzed B-cell lipid rafts by quantitative proteomic analysis,and B-cell raft proteome reveals that ezrin regulates antigenreceptor-mediated lipid raft dynamics. Roumier et al. [207] hasdemonstrated that ezrin, F-actin (see ACTA1; OMIM 102610),and CD43 (OMIM 182160) relocalize to the sides, not thecenter, of the T-cell-(Antigen Presenting Cell) contact area afterformation of immunologic synapse, which suggested that ezrinmay contribute to setting the scaffold between the actin cyto-skeleton and transmembrane proteins facilitating cell–cellinteractions and receptor retention. Using cells from Cd43 −/−

mice, Allenspach et al. [208] observed that ERM proteins moveindependently of the large CD43 mucin. Overexpression of adominant-negative ERM mutant containing the N-terminal 320amino acids of ezrin inhibited the activation-induced movementof CD43 without affecting conjugate formation. The dominant-negative mutant reduced cytokine production but not theexpression of T-cell activation markers. By double staining ofezrin and palladin (OMIM 608092) in several cell lines,Mykkanen et al. [209] have found that the subcellular localizationof ezrin differed between epithelial and smooth muscle cells. Inepithelial cells, such as HeLa, ezrin localized at the cortical actinskeleton and demonstrated little overlap with palladin. However,in intestinal smooth muscle cells, ezrin demonstrated a filamen-tous staining pattern and partial colocalization with palladin. Yuet al. [210] have established highly and poorly metastatic rhab-domyosarcoma cell lines derived from a transgenic mouse modeloverexpressing Hgf/Sf (142409) and deficient for Ink4a/Arf(OMIM 600160), in which skeletal muscle tumors reminiscent ofthose in embryonic rhabdomyosarcoma (OMIM 268210) arisewith very high penetrance and short latency [211]. Yu et al.(2004), then used cDNAmicroarray analysis of these cell lines toidentify a set of genes whose expression was significantly dif-ferent between highly and poorly metastatic cells. Subsequent invivo functional studies revealed that ezrin and Six1 (OMIM601205) have essential roles in determining the metastatic fate ofrhabdomyosarcoma cells. VIL2 and SIX1 expression was en-hanced in human rhabdomyosarcoma tissue, significantly cor-relating with clinical stage [211]. By imaging osteosarcoma cellsin the lungs of mice, Khanna et al. [212] showed that ezrinexpression provides an early survival advantage for cancer cells


that reach the lung. AKT and MAPK3 (OMIM 601795) phos-phorylation and activitywere reducedwhen ezrinwas suppressed.Ezrin-mediated early metastatic survival was partially dependenton the activation ofMAPKbut not AKTand availability of CD44.To define the relevance of ezrin in the biology of metastasisbeyond the founding mouse model, Khanna et al. have examinedezrin expression in dogs that naturally developed osteosarcoma.High ezrin expression in dog tumors was associated with earlydevelopment of metastases. Consistent with this data, they founda significant association between high ezrin expression and pooroutcome in pediatric osteosarcoma patients [212].

13. Acid sphingomyelinase and raft signaling

Acid sphingomyelinase (OMIM 607608) is a lysosomalsphingomyelin phosphodiesterase (EC 3.1.4.12). Stress is believedto activate sphingomyelinase to generate ceramide, which serves asa second messenger in initiating apoptotic response. The firstconclusive evidence for this paradigm was provided by Santanaet al. [213], who showed that lymphoblast from Niemann–Pickpatients failed to respond to ionizing radiation with ceramide ge-neration and apoptosis. Earlier, Suchi et al. [214] have demonstratedthat the metabolic defect in cultured Niemann–Pick disease cells,which lack sphingomyelinase activity, could be corrected byretroviral-mediated transfer of human ASMase cDNA. Targeteddisruption of the Smpd1 gene (human homologue of SMPD1 gene)in transgenic mice was achieved by homologous recombination inembryonic stem cells [215]. Homozygous mice accumulatedsphingomyelin extensively in the reticuloendothelial system ofliver, spleen, bone marrow, and lung, as well as in the brain. Moststrikingly, the ganglionic cell layer of Purkinje cells of thecerebellum degenerated completely, leading to severe impairmentof neuromotor coordination. The picture resembled that of theneurovisceral form of Niemann–Pick disease type A. Horinouchiet al. [216] obtained similar results in Smpd1 knockout mice. Usingdifferent human epithelial cells and primary fibroblasts, Grassmeet al. [217] demonstrated an activation of phospholipase C andASMase in TNF-α (FAS)-mediated hepatocellular apoptosisinduced by Neisseria gonorrhoeae, resulting in the release ofdiacylglycerol and ceramide. Genetic and/or pharmacologic block-ade of ASMase and phosphatidylcholine-specific phospholipase Ccaused inhibition of cellular invasion by the pathogen. Garcia-Barros et al. [23] have studied that endothelial apoptosis is ahomeostatic factor regulating angiogenesis-dependent tumorgrowth. Moreover, microvascular damage regulates tumor cellresponse to radiation at the clinically relevant dose range.

In recent years ASMase has emerged as a biochemicalmediator of stimuli as diverse as ionizing radiation, chemother-apeutic drugs, UVA light, heat, FAS activation, reperfusioninjury, as well as infection with some pathogenic bacteria andviruses [22–25,218–228]. ASMase activity is also crucial fordevelopmental programmed cell death of oocytes [23,24]. Acomprehensive model has been proposed for a role of ASMaseto explain the function ceramide plays in FAS-induced apop-tosis. Upon contacting the relevant stimuli, ASMase translo-cates into and generates ceramide within distinct plasmamembrane “chol-rafts”. The in situ produced ceramide displaces

cholesterol and transforms “chol-raft” to “cer-raft” (CompareFigs. 1, 3, and 4). Fig. 4 shows a scheme representing newmolecular steps occurring upstream of FAS internalizationinvolving rafts. Posttranslational palmitoylation of FAS (A)allows the targeting of the FAS receptor to lipid raft (precisely,“chol-raft”) (B), where following stimulation (for examples,radiation or chemotherapeutic drug) FASL binds to FAS andASMase translocates to the raft and generates ceramide byhydrolysis of SM and transforms “chol-raft” to “cer-raft”, “cer-rafts” coalesces to larger rafts which eventually sequestersFAS–FASL complex and the connection between FAS receptorand actin cytoskeleton occurs via the association of FAS withezrin (C). The ezrin mediated cytoskeleton association initiates“cer-raft”–FAS–FASL internalization (D), a prerequisite stepfor the intracellular optimal formation of DISC (E), which leadsto an efficient caspase activation and cell death (see also refs.[24,199,200]). DeMorrow et al. [222] have recently investi-gated the mechanism of actions of two endocannabinoids,namely, anandamide and 2-arachidonylglycerol, on regulationof cholangiocarcinoma growth. It was observed that althoughanandamide was antiproliferative and pro-apoptotic, 2-arachi-donylglycerol stimulated cholangiocarcinoma cell growth.Interestingly, target of action of both the drugs was lipid rafts.2-arachidonylglycerol treatment effectively dissipated the lipidraft structures and caused the lipid raft-associated proteins lynand flot-1 [1,8,147–149] to disperse into the surrounding mem-brane. Anandamide treatment induced an accumulation ofceramide, which was required for anandamide-induced suppres-sion of cell growth by FAS-mediated apoptosis. Natural inhi-bitors of angiogenesis like, thrombospondin-I and pigmentepithelium-derived factor induce FAS/FASL-mediated apopto-sis and block angiogenesis without harming the preexistingvasculature. Volpert et al. have demonstrated that both inhbitorsupregulated FASL on endothelial cells, thereby specificallysensitizing the stimulated cells to apoptosis by inhibitor-inducedFASL. It may be considered as an example of cooperationbetween pro- and antiangiogenic factors in the inhibition ofangiogenesis, and provided one explanation for the ability ofinhibitors to select remodeling capillaries for destruction[190]. The studies documenting selective effects of lipid rafts forchemotherapeutic induction of apoptosis and cell cycle arrestfor cancer cells were described by Gajate and Mollinedo[153,154,225–227], while investigating the mechanism of actionof the novel anti-tumor drugs edelfosine and aplidin. They andothers have discovered a potent and novel cell-killing mechanismthat involves the formation of FAS-driven scaffolds in membraneraft clusters, housing death receptors and apoptosis-relatedmolecules after application of chemotherapeutic drugs. FAS,tumor necrosis factor-receptor 1 (TNF-R1) and TNF-relatedapoptosis-inducing ligand (TRAIL)-R2/DR5 were clustered intolipid rafts of various cell lines, including leukemic Jurkat, M624melanoma, and breast cancer cells following radiation or drugtreatment, and the presence of FAS had been being essential forapoptosis [22–24,153,154,153,154,219–227]. Actin-linking pro-teins ERM-trio, RhoA and RhoGDI were also clustered into FAS-enriched rafts in drug-treated leukemic cells [153,154,225–228].The functions of SM in FAS-mediated apoptosis by FAS clustering

Fig. 4. A scheme of molecular steps involving raft that occur upstream of FAS internalization Posttranslational palmitoylation of FAS (A) allows the targeting of theFAS receptor to lipid raft (precisely, “chol-raft”) (B) where following stimulation FASL binds to FAS and ASMase translocates to the raft and generates ceramide byhydrolysis of SM and transforms “chol-raft” to “cer-raft”, “cer-rafts” coalesce to larger rafts which eventually sequester FAS–FASL complex and the connectionbetween FAS receptor and actin cytoskeleton occurs via the association of FAS with ezrin (C). The ezrin mediated cytoskeleton association initiates “cer-raft”-receptorinternalization (D), a prerequisite step for the intracellular optimal formation of DISC (E), which leads to an efficient caspase activation and cell death. Modified afteradaptation with permission from Chakrabandhu et al. [200].


through aggregation in lipid rafts have nicely been demonstratedby cloning a gene responsible for SM synthesis (SMS1), andestablishing SM-synthase-defective WR19L cells transfected withthe human Fas gene (WR/Fas–SM(−)), and cells that have beenfunctionally restored by transfection with SMS1 (WR/Fas–SMS1)[224]. It has been observed that production of membrane SMenhances FAS-mediated apoptosis through efficient translocationof FAS into lipid rafts, FAS clustering increasing DISC formation,activation of caspases with concomitant increase in ceramidegenerationwithin lipid rafts upon FAS stimulation [221,224]. FAS-mediated apoptosis involves FAS activation both in a FASL-independent way and following FAS–FASL interaction in anautocrine way, through the accumulation of FAS, membrane-bound FASL and signaling molecules in membrane rafts and highamount of ceramide [23,24,224–228].

14. Discussion and perspectives

Structural and functional role of lipid rafts at the plasmamembrane as well as in cell organelles, including Endoplasmicreticulum and Golgi apparatus has been analyzed and reviewed indetail in several studies [1,3,5,7–17,19–24]. In recent years aspecific activity of membrane sub-domains, including SM,sphingosine and ceramide has been observed to contribute tocell death by apoptosis [19–24,218–222]. Although detected invarious cell types, the role of such sub-domains in apoptosis hashowever been mostly studied in lymphocytes and endothelialcells where the physiological apoptotic program occurs after FAStriggering and in situ generation of sphingosine and ceramide byASMase activity [19–24,218–223]. Current data suggest thatceramide–sphingolipid rich sub-domains mimic cholesterol–

sphingolipid rich lipid rafts, and manipulation of ceramidemetabolism and/or the function of ceramide-enriched membraneplatforms may present novel therapeutic opportunities for thetreatment of cancer, degenerative disorders, pathogenic infectionsor cardiovascular diseases [24,153,154,221–228].

Sphingolipid- and cholesterol enriched membrane microdo-mains are considered to be floating in an “ocean” of phospholipid,and hence have been termed rafts [16]. Although there is disputein the field as to the size of these domains, it is usually safeto say that they range in size from 10 to 200 nm in diameter[9,10,18]. Recent contribution to this field is the recognition thatconsumption of sphingolipids within rafts to generate ceramideresults in dramatic alteration of these small rafts. Ceramide resultsin the coalescence of sub-domains into large-membrane macro-domain. These structures serve as platforms for protein con-centration and oligomerization for transmitting signals acrossthe plasma membrane (see Figs. 1, and 3). Ceramide, which hasthe unique property of fusing membranes and the capability toself-associating through hydrogen bonding (reviewed by vanBlitterswijk et al., [229]), appears to drive the coalescence of raftmicrodomains to form large, ceramide-enriched membraneplatform, which exclude cholesterol [19,20,24,219–221]. Studieswith model membrane, unilamellar vesicles composed ofphosphatidylcholine/sphingomyelin that was locally treatedwith sphingomyelinase, showed that ceramide generation isfollowed by the formation of patches by ceramide that coalescedrapidly into ceramide-enriched macrodomain [19,20,230]. Theformation of distinct domains in model membranes upon theaddition of ceramidewas also demonstrated byHuang et al. [231],who employed a magnetic resonance spectroscopy technique todemonstrate a transition of fluid phospholipid bilayers (non-raft)


into a gel phase (raft-like) upon addition of ceramide. Veiga et al.[232] similarly demonstrated that ceramide partitions into distinctdomains instead of mixing with phospholipids. Displacement ofcholesterol by ceramide from lipid rafts decreases the associationof the raft-binding and cholesterol binding proteins, for example,cav-1 [233].

Outstanding questions arise: where does the displacedcholesterol go? The displaced cholesterol may move to theother parts of the phospholipid ocean. Cellular cholesterol iscontinuously balanced by influx of exogenous cholesterol andefflux of cellular cholesterol. Cholesterol can be taken up from thelipoproteins of circulation by mechanism involving of deso-rption – transfer of cholesterol from the lipoproteins to theexoplasmic leaflet of the plasma membrane bilayer – or byreceptor-mediated uptake (for detailed discussions, see Simonsand Ikonen, [234]). Cellular cholesterol is continuously lost byrelease of cholesterol to circulating lipoproteins. Such a loss canbe quite rapid, up to 0.1% of total cholesterol per minute. Therelease from the plasma membrane can take place by desorptionof cell surface cholesterol into lipoproteins or be induced afterhigh-density lipoprotein binding to membrane receptors. In manycells, including cancer cells mechanism of cholesterol removal isby membrane shedding, a process releasing plasma membranevesicles thatmay be enriched in raft lipids [1,28–32]. Reasonably,cholesterol behaves differently with respect to efflux whether it isin rafts or non-rafts membrane matrix. Raft cholesterol is moreslowly extracted by cyclodextrin and by HDL; hence, non-raftcholesterol is the most likely source for desorptive efflux. Theincrease in cellular cholesterol levels may lead to increaseddeposits of cholesterol esters (CE) in cytoplasmic lipid droplets.As a matter of fact, deposition of cholesterol was detected invarious tumours almost a century ago [235–237], which also iswell over a period of 60 years before the proposition of lipidbilayer membrane structure (reviewed in refs. [1,234,238]). Inerythrocyte membranes, cholesterol has been found to exhibittransbilayer translocation with a half time of about 50 min, so thatefficient efflux may indeed require a specific flippase. ATP-binding cassette transporter ABCA1 has also been found topromote Ca2+-induced translocation of phosphatidyl serine to theexoplasmic leaflet [239], which may favour the release ofphospholipids and cholesterol to HDL. ABCA1 protein localizedto both the plasmamembrane and to the Golgi complex [239] andcould also be involved in the transport of cholesterol form theGolgi to the cell surface, possibly involving rafts [240].

Other interesting questions: are there quantitative differencesin the amount of lipid rafts, or at least lipid contents of rafts, inbetween normal cells and cancer cells? The number of rafts assuch has not been counted and compared so far between normaland cancer cells. But, elevated levels of cholesterol-rich lipid raftsin cancer cells can be correlatedwith apoptosis sensitivity inducedby cholesterol depleting agents and shedding of membranevesicles from plasma membrane (PM), and data on comparisonsof the lipid contents between normal cells and cancer cells arewidely available. Cholesterol deposition in various tumours hasbeen observed since many decades ago [235–237]. For instance,the clear cell form of RCC is known to derive its Histologicappearance from accumulations of glycogen and lipid. It has been

found that the most consistently stored lipid form is CE [241].Gebhard et al. [241] investigated that clear cell cancer (CCC)tissue contains 8-fold higher total cholesterol and 35-fold moreesterified cholesterol than found in normal kidney. CE appears tobe formed intracellularly since it was not membrane-bound andsince oleate was the predominant form, as opposed to linoleate inlipoprotein CEs. Further, Gebhard et al. analyzed that the cho-lesterol in clear cell tumours may not be appeared as a result ofexcessive synthesis from acetate since HMG–CoA reductaseactivity was lower in those cancer tissues than in normal kidney(2.9+0.8 vs. 7.2+1.2 pmol/mg of protein per min). In contrast,intracellular activity of fatty acyl‑coenzyme A: cholesterol acyltransferase (ACAT) was higher in tumour tissue than in normalkidney (2405+546 vs. 1326+301 pmol/mg of protein per20 min) while cytosolic CE activity appeared normal. CE storagein CCR and other cancers may be a result of primary abnormalityin ACAT activity or it would be a reduced release of free cho-lesterol (relative to cell content) with a secondary elevation inACAT activity [235–238,241]. It has been proposed that pro-gressive increases in membrane cholesterol contribute to theexpansion of rafts/caveolae, which may potentiate oncogenicpathways of cell signaling [1,25,27,46,52,71,127,238]. In agrowing tumour the cells need new blood vessels to take upnutrients and clean up offal. Neo-formed blood vessels may alsobecome pathways for metastasis, circulation of cancer cells atdistant organs and tissues from the full-blown tumour (reviewedin ref. [1]). Activated cells shed fragments of their plasma mem-brane into the extracellular milieu, and the processes of sheddingare particularly evident in actively growing tumour cells thatcontinually need shedding of membrane vesicles, in vitro andin vivo. Extracellular membrane vesicle(s) from tumour cells(EMVTCs) derived from selected areas of plasma mem-brane analyzed and appear to be enriched with rafts components:cholesterol, GM1, GM3 and SM, ceramide, and contain surfaceantigens and proteases often present in tumour cells. [31,242–246]. Dahiya et al. [244] have observed that there are differencesin lipid composition in human prostatic adenocarcinoma celllines, and that the SM level was significantly higher in highlymetastatic cells (DU-145 and ND-1) compared with the lowermetastatic variant (LNCaP). van Blitterswijk et al. [245] havefound that the lipid fluidity in purified PM of murine leukemicGRSL cells, as measured by fluorescence polarization, is muchhigher than in PM of normal thymocytes. In contrast, significantdifferences were found between PM and EMVTCs from GRSLcells. In this system a much lower lipid fluidity of the shedEMVTCs was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and SM (9-fold) content, ascompared to the PM.

Several proteases, including MMPs are enriched in EMVTCs/rafts and thought to play a role in tumour cell invasion andmetastasis [31,32,245–247]. Endothelial cell migration andinvasion through ECM are essential for neo-vascularisation, andEMVTCs/rafts enriched with SM and MMP2/MMP9 was foundcapable to increase the process by 3–5 folds [31,248,249]. Additionof purified EMVTCs and SM independently in culture media maypromote the formation of capillary-like structures of cancer cells[31]. Tumour cells produce various cytokines and chemokine that


attract various leukocytes – neutrophils, eosinophils, macrophages,dendritic cells, andmast cells, aswell as lymphocytes– all ofwhichare capable of producing a spectrum of cytokines and cytotoxicmediators including reactive oxygen species, serine and cysteineproteases, MMPs, membrane perforating agents, and solublemediators of apoptosis, such as TNF-α, interleukins andinterferons, IFNs [1,32,130–132,247–249]. In addition to alteringthe local balance of pro-angiogenic factors during melanomadevelopment and in at least human cervical, breast and prostatecarcinogenesis, angiogenesiswas found to be activated inmid stagelesions, prior to the appearance of full-blown tumours (reviewed inrefs. [1,131]). The progression of human tumors involves theMMPfamily. Two particular members of the family, MMP2 and MMP9,seem to have an important role in invasion and metastasis of variedtumors [30,129–134,137–141]. Chetty et al. [250] have discoveredthat down-regulation ofMMP2by adenovirus-mediated delivery ofMMP2 siRNA (Ad-MMP2) reduced spheroid invasion andangiogenesis in vitro, and metastasis and tumor growth in vivo.Ad-MMP2 infection led to the induction of apoptosis in A549 lungcancer cell lines. Ad-MMP2 decreased the content of theantiapoptotic members of the Bcl-2 family proteins and increasedthe content of the pro-apoptotic members of the Bcl-2 family.Furthermore, Ad-MMP2-mediated apoptosis was accompanied byincrease in truncated Bid, release of cytochrome-C and theactivation of caspase-8, -9 and -3. Ad-MMP2 infection causedup-regulation of FAS–FASL and FADD, and Anti-FASL antibodyreversed Ad-MMP2-induced apoptosis. Tissue inhibitor of metal-loproteinases (TIMP)-3, an endogenous inhibitor of MMP2, whichcleaves FASL and activates the FAS–FASL -inducing apoptoticpathway, was increased in Ad-MMP2-treated cells. Adenovirus-mediated expression ofMMP2 siRNA in human lung xenografts invivo resulted in increased FAS, FASL, cleaved Bid and TIMP3.This show that MMP2 inhibition upregulates TIMP3 levels, whichin turn, promotes apoptosis in lung cancer.

Barnhart et al. [251] have demonstrated that despite theirapoptotic potential, human tumors may be refractory to thecytotoxic effects of FASL. Hence, tumour progression may bepromoted by non-apoptotic signals emanating from FAS thatinclude (but are not limited to) activation of NFκB and all threemajor MAPK pathways: ERK1/2, p38 and JNK1/2. Misra et al.[121] have defined a link among TCR, caspases, and the NFκBpathway that occurs in a sequestered lipid raft environment in T-cells. Watabe et al. [252] have demonstrated that caffeic acidphenethyl ester (CAPE), known to inhibit NFκB, induce apop-tosis via FAS-signal activation in human breast cancer MCF-7cells. CAPE activates FAS by a FASL-independent mechanism,induces p53-regulated Bax protein, and activates caspases. Theexpression of dominant-negative c-Jun, which inhibits the JNKsignal, also suppresses CAPE-induced apoptosis, suggestingMAPKs are involved in CAPE-induced apoptosis. The expres-sion of FAS-antisense oligomers significantly suppressed theCAPE-induced activations of JNK and p38 and apoptosis ascompared with FAS sense oligomers. These phenomena areattributable to the inhibition of NFκB by CAPE, as examined bythe effect of a truncated form of IκBα (IκBδN) lacking thephosphorylation sites essential for NFκB activation. IκBδNexpression not only inhibited NFκB activity but also induced

Fas activation, Bax expression, and apoptosis. These findingsdemonstrate that NFκB inhibition is sufficient to induceapoptosis and that FASL-independent activation of FAS playsa role in NFκB inhibition-induced apoptosis in MCF-7 cells.

14.1. Tumour suppression and metastasis promotion duality

A protein that is known to have dual function may exertdistinct function through lipid rafts. Biochemical analyses incultured cells have established that cav-1 is a tumor suppressorgene and a negative regulator of the v-Src, H-ras, protein kinaseA, PKC-isoforms, and Ras-p42/44 MAPK kinase cascadewithin caveolae ([8,46] and references therein). Upon with-drawal of cav-1 signal the rate of cell proliferation wouldovertake the rate of apoptosis, thereby cause higher growth ofthe tumor through lipid raft regulation of PI3K/Akt pathway[1,8,25,46–49,65–71]. Alternatively, cav-1 may also act asmetastasis enhancing protein, which is unlikely to its growthsuppression properties [46,67]. For example, filopodia forma-tion with enhanced metastasis in lung adenocarcinoma isassociated with higher expression of cav-1 [46]. As shown inFig. 2, a concert between lipid rafts and cav-1 function isnecessary for filopodia growth and increase of metastaticpotential of lung adenocarcinoma cells. The picture emergesfrom such data is that the tumour cells use some reversiblemolecular programme for tumour growth and cancer metastasis.Two common strategies for shifting the balance may be in-volved: firstly, altered composition of membrane domains; andsecondly, altered gene transcription. One of the many ways ofaltering membrane domains may be the transition of “chol-raft”to “cer-raft” and inactivation of proteins like, cav-1. Since cav-1is predominantly a “chol-raft” marker, cav-1 may not signalthrough “cer-raft” [233]. This is, likely, being the case with UV-radiation and carcinogen induced tumorigenesis, where formationof ceramide may also play the secondary role for ceramide assecond messenger (for outstanding discussion of ceramide assecond messenger, see [229]). But this hypothesis needs moreexperimental support. The second concept, altered gene tran-scription, may favorably be argued as many raft-componentgenes, including cav-1 are epigenetically regulated by dynamicflux of methyl layer at cytosine-5′C of promoter-CpG-islandsof DNA. CpG-island methylation is one of many epigeneticswitches, and play critical roles in controlling expressions of raftproteins involved in tumour growth and cancer metastasis [1].Some other genes, including CD44, E-cad, EGFR, FAS, MMP 2,MMP 9, and uPAR may be regulated by many epigeneticswitches, including DNA methylation and histone modifications[1,72–74,78–95,191–198,253–255]. Inhibition of histone mod-ification enzyme, histone deacetylase, has been implicated forremodeling lipid rafts [256,257]. Lipid raft proteomics dictatesvarious types of exchanges in between intracellular compartmentsand intercellular cell–cell, guest–host vicinity, including enzymeslike, uPA, MMPs; cytokines and motility factors that modify thesurrounding ECM [1,4,35–37,39–45,128–134,258,259]. Raftassociation is essential for proteins like, H-ras, one of the majorcomponents of oncogenic signals to be transported [1,5,8] to thesite of action to exert its biological activity [5,67,260,261]. Raf-1,


a component of lipid rafts, is a downstream regulator of MAPKpathway (Fig. 3). Because the deregulated overexpression ofmany proteins ofMAPKpathway is frequently seen in a variety ofhuman cancers, modulation of MAP kinases by disruption of“chol-raft” along with the use of some natural compound,therapeutic agents and other inhibitors, including MEK1/2inhibitor PD98059, and the PKC-δ inhibitor, rottlerin, mayprovide novel strategies for the prevention and treatment ofcancer.

One of the oncogenic signals is transmitted by Akt also. Aktactivation up-regulates anti-apoptotic genes such as Bcl-XL andFLIP [199,262–268], and recent studies have suggested thatrafts are involved in Akt activation [71,262,269–272]. In cor-relation with Akt inactivation and Bcl-XL down-regulation,methyl-beta-cyclodextrin (MBCD) induces apoptosis through amitochondrial pathway, including mitochondrial membranepotential change and casp-3 activation. It is intriguing thatEGF administration could not restore Akt activation once rafts/caveolae were disrupted, but cholesterol addition may reactivateAkt in the absence of EGF. Consistent with these phenomena,cholesterol repletion but not EGF administration rescued cellsfrom the apoptosis induced by raft/caveola disruption [273,274].Internalization-defective oncogenic EGFR are known to residein caveolae even after ligand binding [104,108], and this pro-longed EGFR activity in caveolae generates altered signalingpathways such as the enhanced tyrosine phosphorylation of thecaveolae molecules, cav-1 and dynamin [64–66,275]. Assumingthat the macromolecular complexes exhibit reduced diffusion inbiological membranes, Lajoie et al. [276] have reported thatcompetition between the galectin lattice and oligomerized cav-1microdomains for EGFR recruitment regulates EGFR signalingin tumor cells. In mammary tumor cells deficient for Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5), a reduction inEGFR binding to the galectin lattice allows an increased as-sociation with stable cav-1 cell surface microdomains that sup-presses EGFR signaling. Depletion of cav-1 enhances EGFRdiffusion, responsiveness to EGF, and relieves Mgat5 deficiency-imposed restrictions on tumor cell growth. In Mgat5(+/+) tumorcells, EGFR associationwith the galectin lattice reduces first-orderEGFR diffusion rates and promotes receptor interaction with theactin cytoskeleton. Importantly, EGFR association with the latticeopposes sequestration by cav-1, overriding its negative regulationof EGFR diffusion and signaling. Therefore, they concluded thatcav-1 is a conditional tumor suppressor whose loss is advanta-geous when beta1,6GlcNAc-branched N-glycans are below athreshold for optimal galectin lattice formation [276], whichprovides a rational supportive explanation for dual function ofcav-1, and inactivation of cav-1 in “cer-raft” enriched membranedomains [233]. A construct of B82L cells transfected with non-internalizing oncogenic EGFR underwent apoptosis after MBCDtreatment indicating that the cholesterol depletion can induceapoptosis in oncogenic pathways involving EGFR inactivation[262]. FAS-, caspase 8-, and caspase 3-dependent signaling issuggested to fold properly the raft assembly by separating non-raftlipids, including aminophospholipid and phosphatidylserine,through activation of aminophospholipid translocase and phos-phatidylserine externalization in human erythrocytes [277].

Molecular modeling with tea polyphenol EGCG and uPA hasrevealed that, EGCG blocks urokinase activity by interacting withH57 and S195 extending toward R35 from a positively charged loopof urokinase, which thus prevents the formation of the catalytictriad of urokinase [123,129,142]. Interestingly, Adachi et al. [278]have demonstrated in HT29 colon cancer cells that, EGCG canalter the lipid order of the membrane during EGFR inhibitoryactivity of EGCG. Their findings suggest that EGCG inhibits thebinding of EGF to the EGFR by altering membrane raftorganization, which prevents the EGFR dimmer formation. Italso may explain the ability of EGCG and similar phytochemicalto inhibit activation of other membrane associated RTKs, and thatthey may play a critical role in the anticancer effects [278].

Finally, the picture becomes clear that there are two types offunctional rafts in living cells: rafts enriched with cholesterol–sphingomyeline–ganglioside, recruit proteins like caveolins,CD44, and members of the RTKs family, designated as “chol-rafts” (Fig. 1, upper portion); and rafts enriched with ceramide–sphingomyeline, prefer proteins like FAS, FASL, Ezrin, andother members of FAS–DISC, designated as “cer-rafts” (Fig. 1,lower portion). “Chol-rafts” are responsible for cellular home-ostasis, but when normal cellular signaling is dysregulated“chol-rafts” promote cell transformation, tumor progression,and metastasis (See Fig. 3); and “cer-rafts” promote endocytosisof the FAS–DISC leading to apoptosis, but when dysregulated“cer-rafts” may also contribute to cell transformation, tumorprogression, and metastasis. Small rafts, either “chol-raft” or“cer-raft” may sometimes be stabilized to form larger platformsthrough clustering of proteins, and be endocytosed by LPLRreordering in living cells to extinguish the specific signals.Elevated cholesterol induces also coalescence of rafts, whichserve to sequester proteins, which are activated after receivingstimuli, including CD44, EGFR, Ras and stimulate “start” sig-nals to various cellular pathways, including tumorigenesis.When cells and tumors are exposed to radiation or challengedwith therapeutic compounds ASMase becomes activated. Theactivated ASMase then translocates to membrane surfaces anddrags more and more SM near in the vicinity (Figs. 1, and 3) andhydrolyzes SM generating sphingosine and ceramide. This insitu produced ceramide rapidly displaces cholesterol frommembrane/lipid-“chol-raft” and generates “cer-raft”. Thesenewly formed “cer-rafts” cause endocytosis of the FAS–DISCand helper proteins, which immediately extinguish the signalsto death/apoptosis (See Fig. 3, and 4).

Here, I have given an overview of how lipid rafts are involvedin cell signaling either for proliferation or for apoptosis. Signalsfor controlled or enhanced proliferation are transduced viacaveolae protein, cav-1 and “chol-raft”-containing proteins ofthe RTKs family (for example, EGFR); whereas signals forapoptosis are transduce through “cer-raft”-containing proteins ofthe FAS–DISC (for example, FAS itself). The single mostnotable feature of rafts is their participation in somany aspects ofcell biology, ranging from the fundamental (for example, cellpolarity) to the highly specialized (for examples, angiogenesisand immune escape). In the future there will no doubt be manymore surprises and examples of cellular controls by these mole-cular assemblages. The analyses of individual raft proteome of


lipid rafts have provided numerous instances where lipid raftsinfluence the signaling function of other cell membrane re-ceptors. Lipid rafts can either inhibit or activate other rafts, insome cases this results in intrinsic modulation of cell fate. Theseeffects may account for the potency of lipid rafts antagonists forinhibiting angiogenesis. Lipid rafts most often endocytosed withgrowth factor as well as death-receptor in signaling pathways toenhance their respective activity. Detailed analysis of the MAPkinase and FAS pathways has revealed multiple points of inter-sections, suggesting a complex network of interactions coordi-nating cell survival and death. The importance of cytoskeletalorganization and mechanical tension for raft mediated signalingthereby provides a mechanism by which these considerationscan influence the responses to growth factors as well, providing aconvergence point for different classes of extracellular cues.Lipid rafts can also cause ligand-independent trans-activation oftyrosine kinase growth factor receptors like, EGFR, and deathreceptors like, FAS. Effects of this type have the potential tobroaden the range of rafts signals but, when uncontrolled, resultin tumorigenesis.

The picture emerging from these studies is that lipid raftsfunction as nodes within webs of signaling, adhesive, cyto-skeletal, proliferative and apoptotic pathways. These complexnetworks enable lipid raft-mediated participation of individualproteins in highly regulated processes such as migration, growthcontrol and morphogenesis. But like all systems greater com-plexity also affords greater opportunities for subversion. Under-standing these circuits in more detail should shed light on avariety of pathologic states; including cancer, impaired immunefunction, atherosclerosis, Parkinson's disease, as well as wouldshed light on intrinsic robustness of homeostasis.

Acknowledgement

I apologise for many other important contributions that I havenot been able to include and discuss.

References

[1] S.K. Patra, S. Bettuzzi, Epigenetic DNA methylation regulation of genescoding for lipid raft-associated components: a role for raft proteins in celltransformation and cancer progression (Review), Oncol. Rep. 17 (2007)1279–1290.

[2] A.M. Davey, R.P. Walvick, Y. Liu, A.A. Heikal, E.D. Sheets, Membraneorder and molecular dynamics associated with IgE receptor cross-linkingin mast cells, Biophys. J. 92 (2007) 343–355.

[3] K. Jacobson, O.G. Mouritsen, R.G. Anderson, Lipid rafts: at a crossroadbetween cell biology and physics, Nat. Cell Biol. 9 (2007) 7–14.

[4] N. Gupta, B. Wollscheid, J.D. Watts, B. Scheer, R. Aebersold, A.L.DeFranco, Quantitative proteomic analysis of B cell lipid rafts revealsthat ezrin regulates antigen receptor-mediated lipid raft dynamics, Nat.Immunol. 7 (2006) 625–633.

[5] K. Simons, D. Toomre, Lipid rafts and signal transduction, NatureReviews / Mol. Cell Biol. 1 (2000) 31–41.

[6] M. Dykstra, A. Cherukuri, H.W. Sohn, S.J. Tzeng, S.K. Pierce, Locationis everything: lipid rafts and immune cell signaling, Annu. Rev. Immunol.21 (2003) 457–481.

[7] S.K. Patra, A. Alonso, F.M. Goni, Detergent solubilizations ofphospholipid bilayer in the gel state: the role of polar and hydrophobicforces, Biochim. Biophys. Acta. 1373 (1998) 112–118.

[8] F. Galbiati, B. Razani, M.P. Lisanti, Emerging themes in lipid rafts andcaveolae, Cell 106 (2002) 403–411.

[9] R. Varma, S. Mayor, GPI-anchored proteins are organized in submicrondomains at the cell surface, Nature 394 (1998) 798–801.

[10] P. Sharma, R. Varma, R.C. Sarasij, K. Ira–Gousset, G. Krishnamoorthy,M. Rao, S. Mayor, Nanoscale organization of multiple GPI-anchoredproteins in living cell membranes, Cell 116 (2004) 77–589.

[11] S.K. Patra, A. Alonso, J.L.R. Arrondo, F.M. Goni, Liposome containingsphingomyelin and cholesterol: detergent solubilization and infraredspectroscopic studies, J. Liposome Res. 9 (1999) 247–260.

[12] M.I. Collado, F.M. Goni, A. Alonso, D. Marsh, Domain formation insphingomyelin/cholesterol mixed membranes studied by spin-label electronspin resonance spectroscopy, Biochemistry 44 (2005) 4911–4918.

[13] F.R. Maxfield, Plasma membrane microdomains, Curr. Opin. Cell Biol.14 (2002) 483–487.

[14] D.A. Brown, E. London, Structure and function of sphingolipids- andcholesterol-richmembrane rafts, J. Biol. Chem. 275 (2000) 17221–17224.

[15] K. Simons, R. Enhalt, Cholesterol, lipid rafts and disease. Perspective onBiol. and Biochem. of Cholesterol, J. Clin. Invest. 110 (2002) 597–603.

[16] K. Simons, E. Ikonen, Functional rafts in cell membrane, Nature. 387(1997) 569–572.

[17] M. Edidin, The state of lipid rafts: from model membranes to cells, Annu.Rev. Biophys. Biomol. Struct. 32 (2003) 257–283.

[18] L.J. Pike, Rafts defined: a report on the Keystone symposium on lipidrafts and cell function, J. Lipid Res. 47 (2006) 1597–1598.

[19] M. London, E. London, Ceramide selectively displaces cholesterol fromordered lipid domain (rafts), J. Biol. Chem. 279 (2004) 9997–10004.

[20] J.M. Holopainen, M. Subramanian, P.K. Kinnunen, Sphingomyelinaseinduces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane, Biochemistry 37 (1998) 17562–17570.

[21] P. Gopalakrishna, N. Rangaraj, G. Pande, Cholesterol alters the interactionof glycosphingolipidGM3with alpha5beta1 integrin and increases integrin-mediated cell adhesion to fibronectin, Exp. Cell Res. 300 (2004) 43–53.

[22] J.A. Rotolo, J. Zhang, M. Donepudi, H. Lee, Z. Fuks, R. Kolesnick,Caspase-dependent and -independent activation of acid sphingomyeli-nase signaling, J. Biol. Chem. 280 (2005) 25434–26425.

[23] M. Garcia-Barros, F. Paris, C. Cordon-Cardo, D. Lyden, S. Rafii, A.Haimovitz-Friedman, Z. Fuks, R. Kolesnick, Tumor response to radiotherapyregulated by endothelial cell apoptosis, Science 300 (2003) 1155–1159.

[24] E. Gulbins, R. Kolesnick, Raft ceramide in molecular medicine,Oncogene 22 (2003) 7070–7077.

[25] M.R. Freeman, B. Cinar, M.L. Lu, Membrane rafts as potential sites ofnongenomic hormonal signaling in prostate cancer, Trends Endocrin.Metabol. 16 (2005) 273–279.

[26] L.A. Carver, J.E. Schnitzer, R.G.W. Anderson, S. Mohla, Role ofcaveolae and lipid rafts in cancer: workshop summary and future needs,Cancer Res. 63 (2003) 6571–6574.

[27] F.R. Maxfield, I. Tabas, Role of cholesterol and lipid organisation indisease, Nature 438 (2005) 612–621.

[28] C. Gil, R. Cubi, J. Aguilera, Shedding of the p75(NTR) neurotrophinreceptor is modulated by lipid rafts, FEBS Lett. 581 (2007) 1851–1858.

[29] P. Janich, D. Corbeil, GM(1) and GM(3) gangliosides highlight distinctlipid microdomains within the apical domain of epithelial cells, FEBSLett. 581 (2007) 1783–1787.

[30] W. Deng, R. Li, S. Ladisch, Influence of cellular ganglioside depletion ontumour formation, J. Natl. Cancer Inst. 92 (2000) 912–917.

[31] C.W. Kim, H.M. Lee, T.H. Lee, C. Kang, H.K. Kleinman, Y.S. Gho,Extracellular membrane vesicles from tumour cells promote angiogenesisvia sphingomyelin, Cancer Res. 62 (2002) 6312–6317.

[32] V. Dolo, S. D'Ascenzo, S. Violini, L. Pompucci, C. Festuccia, A. Ginestra,M.L. Vittorelli, S. Canevari, A. Pavan, Matrix degraded proteinases areshed in membrane vesicles by ovarian cancer cells in vivo and in vitro,Clin. Exp. Metastasis 17 (1999) 31–140.

[33] G. van Meer, The different hues of lipid rafts. Perspectives on cellbiology. Science 296 (2002) 855–857.

[34] D.A. Zacharias, J.D. Violin, A.C. Newton, R.Y. Tsien, Partitioning oflipid modified monomers GFPs into membrane microdomain of live cells,Science 296 (2002) 913–916.


[35] T. Nebl, K.N. Pestonjamasp, J.D. Leszyk, J.L. Crowley, S.W. Oh, E.J. Luna,Proteomic analysis of a detergent-resistant membrane skeleton fromneutrophil plasma membranes, J. Biol. Chem. 277 (2002) 43399–43409.

[36] L.J. Foster, C.L. de Hoog, M. Mann, Unbiased quantitative proteomics oflipid rafts shows high specificity for signaling factors, Proc. Natl. Acad.Sci. 100 (2003) 813–5818.

[37] M. Gronborg, T.Z. Kristiansen, A. Iwahori, R. Chang, R. Reddy, N.Sato, H. Molina, O.N. Jensen, R.H. Hruban, M.G. Goggins, A. Maitra,A. Pandey, Biomarker discovery from pancreatic cancer secretomeusing a differential proteomic approach, Mol. Cell. Proteomics 5 (2006)57–171.

[38] D. Lichtenberg, F.M. Goni, H. Heerklotz, Detergent-resistant membranesshould not be identified with membrane rafts, Trends Biochem. Sci. 30(2005) 430–436.

[39] Y.H. Say, N.M. Hooper, Contamination of nuclear fractions with plasmamembrane lipid rafts, Proteomics 7 (2007) 1059–1064.

[40] P.A. Everley, J. Krijgsveld, B.R. Zetter, S.P. Gygi, Quantitative cancerproteomics: stable isotope labeling with amino acids in cell culture(SILAC) as a tool for prostate cancer research, Mol. Cell. Proteomics 3(2004) 729–735.

[41] P. Mannova, R. Fang, H. Wang, B. Deng, M.W. McIntosh, S.M. Hanash,L. Beretta, Modification of host lipid raft proteome upon hepatitis C virusreplication, Mol. Cell. Proteomics 5 (2006) 2319–2325.

[42] S. Lacour, A. Hammann, S. Grazide, D. Lagadic-Gossmann, D. Athias,O. Sergent, G. Laurent, P. Gambert, E. Solary, M.T. Dimanche-Boitrel,Cisplatin-induced CD95 redistribution into membrane lipid rafts of HT29human colon cancer cells, Cancer Res. 64 (2004) 3593–3598.

[43] A. Pullikuth, E. McKinnon, H.-J. Schaeffer, A.D. Catling, The MEK1scaffolding protein MP1 regulates cell spreading by integrating PAK1 andRho signals, Mol. Cell. Biol. 25 (2005) 5119–5133.

[44] D. Chakraborty, S. Banerjee, A. Sen, K.K. Banerjee, P. Das, S. Roy,Leishmania donovani affects antigen presentation of macrophage bydisrupting lipid rafts, J. Immunol. 175 (2005) 3214–3224.

[45] A.I. Nesvizhskii, R. Aebersold, Interpretation of shotgun proteomicdata: the protein inference problem, Mol. Cell. Proteomics 4 (2005)1419–1440.

[46] C.-C.Ho, P.-H.Huang,H.-Y.Huang,Y.-H. Chen, P.-C.Yang, S.-M.Hsu,Up-regulated caveolin-1 accentuates the metastasis capability of lung adeno-carcinoma by inducing filopodia formation, Am. J. Pathol. 161 (2002)1647–1656.

[47] K.Wiechen, L. Diatchenko, A. Agoulink, K.M. Scharff, H. Schober, K. Arlt,B. Zhumabayeva, P.D. Siebert, M. Dietel, R. Schafer, C. Sers, Caveolin-1 isdown regulated in human ovarian carcinoma and acts as candidate tumoursuppressor gene, Am. J. Pathol. 159 (2001) 1635–1643.

[48] S. Seveau, R. Eddy, F.R. Maxfield, L. Pierini, Cytoskeleton dependentmembrane domain segregation during neutrophil polarization, Mol. Biol.Cell 12 (2001) 3550–3562.

[49] C. Gomez-Mouton, J.L. Abad, E. Mira, R.A. Lacalle, E. Gallardo, S.Jimenez-Baranda, I. Illa, A. Bernad, S. Manes, C. Martinez-A, Segregationof leading-edge and uropod components into specific lipid rafts duringTcellpolarization, Proc. Natl. Acad. Sci. 98 (2001) 9642–9647.

[50] I. Zucchi, A. Prinetti, M. Scotti, V. Valsecchi, R. Valaperta, E. Mento, R.Reinbold, P. Vezzoni, S. Sonnino, A. Albertini, R. Dulbecco, Associationof rat8 with Fyn protein kinase via lipid rafts is required for rat mammarycell differentiation in vitro, Proc. Natl. Acad. Sci. 101 (2004) 1880–1885.

[51] Q. Zhang, K. Furukawa, H.H. Chen, R. Fujinawa, Y. Kozutsumi, A.Suzuki, T. Urano, K. Furukawa, Down-regulation of caveolin-1 in mouseLewis lung cancer P29 is a causal factor for the malignant properties in ahigh-metastatic subline, Oncol. Rep. 16 (2006) 289–294.

[52] M.R. Freeman, B. Cinar, J. Kim, N.K. Mukhopadhyay, D. Di Vizio, R.M.Adam, K.R. Solomon, Transit of hormonal and EGF receptor-dependentsignals through cholesterol-rich membranes, Steroids 72 (2007)210–217.

[53] J.-P. Gratton, P. Bernatchez, W.C. Sessa, Caveolae and caveolins in thecardiovascular system, Circ. Res. 94 (2004) 1408–1417.

[54] A. Wittenbogaard, E. Smart, Palmitoylation of caveolin-1 is required forcholesterol binding, chaperon complex formation and rapid transport ofcholesterol to caveolae, J. Biol. Chem. 275 (2000) 5595–25599.

[55] D.R. Wood, J.S. Nye, N.J. Lamb, A. Fernandez, M. Kitzmann, Intra-cellular retention of caveolin-1 in presenilin-deficient cells, J. Biol. Chem.280 (2005) 6663–6668.

[56] M. Drab, P. Verkade, M. Elger, M. Kasper, M. Lohn, B. Lauterbach, J.Menne, C. Lindschau, F. Mende, F.C. Luft, A. Schedl, H. Haller, T.V.Kurzchalia, Loss of caveolae, vascular dysfunction, and pulmonary defectsin caveolin-1 gene-disrupted mice, Science 293 (2001) 2449–2452.

[57] B. Razani, J.A. Engelman, X.B. Wang, W. Schubert, X.L. Zhang, C.B.Marks, F. Macaluso, R.G. Russell, M. Li, R.G. Pestell, D. Di Vizio, H.Hou Jr., B. Kneitz, G. Lagaud, G.J. Christ, W. Edelmann, M.P. Lisanti,Caveolin-1 null mice are viable but show evidence of hyperproliferativeand vascular abnormalities, J. Biol. Chem. 276 (2001) 38121–38138.

[58] J. Yu, S. Bergaya, T. Murata, I.F. Alp, M.P. Bauer, M.I. Lin, M. Drab, T.V.Kurzchalia, R.V. Stan,W.C. Sessa, Direct evidence for the role of caveolin-1and caveolae in mechanotransduction and remodeling of blood vessels,J. Clin. Invest. 116 (2006) 1284–1289.

[59] S.K. Patra, M.K. Pal, Spectroscopic probe of the individual and combinedeffect of Triton-X-100 and chloroform on human and bovine serumalbumins and serum albumin–bilirubin complexes, Eur. J. Biochem. 246(1997) 658–664.

[60] W. Schubert, F. Sotgia, A.W. Cohen, F. Capozza, G. Bonuccelli, C.Bruno, C. Minetti, E. Bonilla, S. Dimauro, M.P. Lisanti, Caveolin-1(−/−)-and caveolin-2(−/−)-deficient mice both display numerous skeletalmuscle abnormalities, with tubular aggregate formation, Am. J. Pathol.170 (2007) 316–333.

[61] Y.-Y. Zhao, Y. Liu, R.-V. Stan, L. Fan, Y. Gu, N. Dalton, P.-H. Chu, K.Peterson, J. Ross Jr., K.R. Chien, Defects in caveolin-1 cause dilatedcardiomyopathy and pulmonary hypertension in knockout mice, Proc.Nat. Acad. Sci. 99 (2002) 11375–11380.

[62] M.I. Lin, J. Yu, T. Murata, W.C. Sessa, Caveolin-1-deficient mice haveincreased tumor microvascular permeability, angiogenesis, and growth,Cancer Res. 67 (2007) 2849–2856.

[63] M.A. Fernandez, C. Albor, M. Ingelmo-Torres, S.J. Nixon, C. Ferguson,T. Kurzchalia, F. Tebar, C. Enrich, R.G. Parton, A. Pol, Caveolin-1 isessential for liver regeneration, Science 313 (2006) 1628–1632.

[64] K.K. Wary, A. Mariotti, C. Zurzolo, F.G. Giancotti, A requirement forcaveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth, Cell 94 (1998) 625–634.

[65] J.A. Engelman, X. Zhang, F. Galbiati, D. Volonte, F. Sotgia, R.G. Pestell,C. Minetti, P.E. Scherer, T. Okamoto, M.P. Lisanti, Molecular genetics ofthe caveolin gene family: implications for human cancers, diabetes,Alzheimer disease, and muscular dystrophy, Am. J. Hum. Genet. 63(1998) 1578–1587.

[66] W. Zhang, B. Razani, Y. Altschuler, B. Bouzahzah, K.E. Mostov, R.G.Pestell, M.P. Lisanti, Caveolin-1 inhibits epidermal growth factorstimulated lamellipod extension and cell migration in metastaticmammary adenocarcinoma cells (MTLn3): transformation suppressoreffects of adenovirus-mediated gene delivery of caveolin-1, J. Biol.Chem. 275 (2000) 20717–20725.

[67] J. Hulit, T. Bash, M. Fu, F. Galbiati, C. Albanese, D.R. Sage, A. Schiegel,J. Zhurinski, M. Shtutman, A. Ben-Ze'ev, M.P. Lisanti, R.G. Pestell, Thecyclin D-1 gene is transcriptionally repressed by caveolin-1, J. Biol.Chem. 275 (2000) 21209–22203.

[68] G. Yang, L.D. Truong, T.L. Timme, C. Ren, T.M. Wheeler, S.H. Park, Y.Nasu, C.H. Bangma, M.W. Kattan, P.T. Scardino, T.C. Thompson,Elevated expression of caveolin is associated with prostate and breastcancer, Clin. Cancer Res. 4 (1998) 1873–1880.

[69] S.A. Tahir, G. Yang, S. Ebara, T.L. Timme, T. Satoh, L. Li, A. Goltsov, M.Ittmann, J.D. Morrisett, T.C. Thompson, Secreted caveolin-1 stimulatescell survival/clonal growth and contributes to metastasis in androgen-insensitive prostate cancer, Cancer Res. 61 (2001) 3882–3885.

[70] T.C. Thompson, Metastasis related genes in prostate cancer: the role ofcaveolin-1, Cancer Metastasis. Rev. 17 (1999) 439–442.

[71] L. Zhuang, J. Lin, M.L. Lu, K.R. Solomon, M.R. Freeman, Cholesterol-rich lipid rafts mediate Akt-regulated survival in prostate cancer cells,Cancer Res. 62 (2002) 2227–2231.

[72] Patra, S.K., Patra, A., Rizzi, F., Ghosh, T.C., Bettuzzi, S., 2007, CpG-sequence binding proteinMBD2, active demethylation of intact (cytosine-


5-C-methyl) DNA and regulation of transcription — The state of the art.(Manuscript submitted).

[73] J. Cui, L.R. Rohr, G. Swanson, V.O. Speights, T. Maxwell, A.R. Brothman,Hypermethylation of the caveolin-1 gene promoter in prostate cancer,Prostate 46 (2001) 249–256.

[74] J. Liu, M. Ikeguchi, S. Nakamura, N. Kaibara, Re-expression of thecadherin–catenin complex in lymph nodes with metastasis in advancedgastric cancer: the relationship with patient survival, J. Exp. Clin. CancerRes. 21 (2002) 65–71.

[75] N. Foger, R. Marhaba, M. Zoller, Involvement of CD44 in cytoskeletonrearrangement and raft reorganization in T cells, J. Cell Sci. 114 (2000)1169–1178.

[76] S. Oliferenko, K. Paiha, T. Harder, V. Gerke, C. Schwarzler, H. Schwarz,H. Beug, U. Gunthert, L.A. Huber, Analysis of CD44 containing lipidrafts: recruitment of Annexin II and stabilization by the actin cytoskeleton,J. Cell Biol. 146 (1999) 843–854.

[77] S. Oliferenko, I. Kaverina, J.V. Small, L.A. Huber, Hyaluronic acid (HA)binding to CD44 activates Rac1 and induces lamllipodia out growth,J. Cell Biol. 148 (2000) 1159–1164.

[78] H. Kito, H. Suzuki, T. Ichikawa, N. Sekita, N. Kamia, K. Akakura, T.Igarashi, T. Nakayama, M. Watanabe, K. Harigaya, H. Ito, Hypermethy-lation of the CD44 gene is associated with progression and metastasis ofhuman prostate cancer, Prostate 49 (2001) 110–115.

[79] W. Lou, D. Krill, R. Dhir, M.J. Becich, J.-T. Dong, H.F. Frierson Jr., W.B.Isaacs, J.T. Isaacs, A.C. Gao, Methylation of CD44 metastasis suppressorgene in human prostate cancer, Cancer Res. 59 (1999) 2329–2331.

[80] M. Hasegawa, H.H. Nelson, E. Peters, E. Ringstrom, M. Posner, K.T.Kelsey, Patterns of gene promoter methylation in squamous cell cancer ofthe head and neck, Oncogene 21 (2002) 4231–4236.

[81] A. Bankfalvi,M.KraBort, I.B. Buchwalow,A. Vegh, E. Felszeghy, J. Piffko,Gains and loses of adhesion molecules (CD44, E-Cadherin, and b-Catenin)during oral carcinogenesis and tumour progression, J. Pathol. 198 (2002)343–351.

[82] G.F. Weber, R.T. Bronson, J. Ilagan, H. Cantor, R. Schmits, T.W. Mak,Absence of the CD44 gene prevents sarcoma metastasis, Cancer Res. 62(2002) 2281–2286.

[83] N.S. Verkaik, J. Trapman, J.C. Romijn, T.H. Van Dyer Kwast, G.J. vanSteenbrugge, Down-regulation of CD44 expression in human prostaticcarcinoma cell lines is correlated with DNA hypermethylation, Int.J. Cancer 80 (1999) 439–443.

[84] P. Kogerman, M.-S. Sy, L.A. Culp, Counter-selection for over expressedhuman CD44s primary tumour versus lung metastases in mouse fibro-sarcoma model, Oncogene 15 (1997) 1407–1416.

[85] H. Shiratori, T. Koshino, M. Uesugi, H. Nitto, T. Saito, Acceleration oflung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice, Cancer Lett. 170 (2001) 177–182.

[86] S.K. Patra, A. Patra, H. Zhao, R. Dahiya, DNA methyltransferase anddemethylase in human prostate cancer,Mol. Carcinog. 33 (2002) 163–171.

[87] S.K. Patra, A. Patra, H. Zhao, P. Carroll, R. Dahiya, Methyl-CpG–DNAbinding proteins in human prostate cancer: expression of CXXC sequencecontainingMBD1 and repression ofMBD2 andMeCP2, Biochem.Biophys.Res. Commun. 302 (2003) 759–766.

[88] L.A. Ribeiro-Filho, J. Franks, M. Sasaki, H. Shiina, L.-C. Li, D. Nojima,S. Arap, P. Carroll, H. Enokida, M. Nakagawa, S. Yonezawa, R. Dahiya,CpG hypermethylation of promoter region and inactivation of E-cadheringene in human bladder cancer, Mol. Carcinog. 34 (2002) 187–198.

[89] H. Karube, H. Masuda, Y. Ishii, T. Takayama, E-Cadherin expression isinversely proportional to tumour size in experimental liver metastasis,J. Surg. Res. 106 (2002) 173–178.

[90] M.L. Alpaugh, J.S. Tomlinson, S. Kasraeian, S.H. Barsky, Cooperative roleof E-Cadherin sialyl-Lewis X/A-deficient MUC1 in the passive dissemina-tion of tumour emboli in inflammatory breast carcinoma, Oncogene 21(2002) 3631–3643.

[91] M. Ikeguchi,M.Makino, N. Kaibara, Clinical significance of E-cadherin–catenin complex expression in metastatic foci of colorectal carcinoma,J. Surg. Oncol. 77 (2001) 201–207.

[92] S. Kase, K. Sugio, K. Yamazaki, T. Okamoto, T. Yano, K. Sugimachi,Expression of E-Cadherin and β-Catenin in human non-small cell

lung cancer and the clinical significance, Clin. Cancer Res. 6 (2000)4784–4796.

[93] W.G. Jiang, R.E. Mansel, E-cadherin complex and its abnormalities inhuman breast cancer, Surg. Oncol. 9 (2000) 151–171.

[94] C.G. Kleer, K.L. van Golen, T. Braun, S.D. Merajver, Persistant E-cadherin expression in inflammatory breast cancer, Mod. Pathol. 14 (2001)458–464.

[95] L.R. Cavalli, C.A. Urban, D. Dai, S. de Assis, D.C. Tavares, J.D. Rone,L.F. Bleggi-Torres, R.S. Lima, I.J. Cavalli, J.P. Issa, B.R. Haddad,Genetic and epigenetic alterations in sentinel lymph nodes metastaticlesions compared to their corresponding primary breast tumors, CancerGenet. Cytogenet. 146 (2003) 33–40.

[96] G. Carpenter, S. Cohen, Epidermal growth factor, Ann. Rev. Biochem. 48(1979) 193–216.

[97] M.S. Urdea, J.P. Merryweather, G.T. Mullenbach, D. Coit, U. Heberlein,P. Valenzuela, P.J. Barr, Chemical synthesis of a gene for human epi-dermal growth factor urogastrone and its expression in yeast, Proc. Nat.Acad. Sci. 80 (1983) 7461–7465.

[98] G. Carpenter, The EGF receptor: a nexus for trafficking and signaling,Bioessays 22 (2000) 697–707.

[99] R.N. Jorissen, F. Walker, N. Pouliot, T.P. Garrett, C.W. Ward, A.W.Burgess, EGF receptor: mechanisms of activation and signalling, Exp.Cell Res. 284 (2003) 31–53.

[100] K. Helin, T. Velu, P. Martin, W.C. Vass, G. Allevato, D.R. Lowy, L.Beguinot, The biological activity of the human epidermal growth factorreceptor is positively regulated by its C-terminal tyrosines, Oncogene 6(1991) 825–832.

[101] N. Yang, Y. Huang, J. Jiang, S.J. Frank, Caveolar and lipid raftlocalization of the growth hormone receptor and its signaling elements:impact on growth hormone signaling, J. Biol. Chem. 279 (2004)20898–20905.

[102] M. Yamabhai, R.G. Anderson, Second cysteine-rich region of epidermalgrowth factor receptor contains targeting information for caveolae/rafts,J. Biol. Chem. 277 (2002) 24843–24846.

[103] M.G. Wagh, D. Lawson, J.J. Hsuan, Epidermal growth factor receptoractivation is localized within low-buoyant density, non-caveolar mem-brane domains, Biochem. J. 337 (1999) 591–597.

[104] C. Puri, D. Tosoni, R. Comai, A. Rabellino, D. Segat, F. Caneva, P. Luzzi, P.P.Di Fiore, C. Tacchetti, Relationships between EGFR-signaling-competentand endocytosis-competent membrane microdomains, Mol. Biol. Cell 16(2005) 2704–2718.

[105] D.S. Salomon, R. Brandt, F. Ciardiello, N. Normanno, Epidermal growthfactor-related peptides and their receptors in human malignancies, Crit.Rev. Oncol. Hematol. 19 (1995) 183–232.

[106] A. Wilde, E.C. Beattie, L. Lem, D.A. Riethof, S.H. Liu, W.C. Mobley, P.Soriano, F.M. Brodsky, EGF receptor signaling stimulates SRC kinasephosphorylation of clathrin, influencing clathrin redistribution and EGFuptake, Cell 96 (1999) 677–687.

[107] E.S. Kim, F.R. Khuri, R.S. Herbst, Epidermal growth factor receptorbiology (IMC-C225), Curr. Opin, Oncol. 13 (2001) 508–513.

[108] A. Balbis, A. Parmar, Y. Wang, G. Baquiran, B.I. Posner, Compartmen-talization of signaling-competent epidermal growth factor receptors inendosomes, Endocrinology 148 (2007) 2944–2954.

[109] S.D. Conner, S.L. Schmid, Regulated portals of entry into the cell, Nature422 (2003) 37–44.

[110] L. Lanzetti, V. Rybin, M.G. Malabarba, S. Christoforidis, G. Scita, M.Zerial, P.P. Di Fiore, The Eps8 protein coordinates EGF receptor signallingthrough Rac and trafficking through Rab5, Nature 408 (2000) 374–377.

[111] J.J. Gildea, M.A. Harding, M. Jabed Seraj, K.M. Gulding, D.Theodorescu, The role of Ral A in epidermal growth factor receptor-regulated cell motility, Cancer Res. 62 (2002) 982–985.

[112] J.S. de Jong, P.J. van Diest, P. van der Valk, J.P. Baak, Expression of growthfactors, growth-inhibiting factors, and their receptors in invasive breastcancer. II: correlations with proliferation and angiogenesis, J. Pathol. 184(1998) 53–57.

[113] D.M. Shin, J.Y. Ro, W.K. Hong, W.N. Hittelman, Dysregulation ofepidermal growth factor receptor expression in multistep process of headand neck tumorigenesis, Cancer Res. 54 (1994) 3153–3159.


[114] R. Radinsky, S. Risin, D. Fan, Z. Dong, D. Bielenberg, C.D. Bucana, I.J.Fidler, Level and function of epidermal growth factor receptor predict themetastatic potential of human colon carcinoma cells, Clin. Cancer Res. 1(1995) 19–31.

[115] M. Nagane, F. Coufal, H. Lin, O. Bögler, W.K. Cavenee, H.J. Huang, Acommon mutant epidermal growth factor receptor confers enhancedtumorigenicity on human glioblastoma cells by increasing proliferationand reducing apoptosis, Cancer Res. 56 (1996) 5079–5086.

[116] S.M. Thomas, N.E. Bhola, Q. Zhang, S.C. Contrucci, A.L. Wentzel, M.L.Freilino, W.E. Gooding, J.M. Siegfried, D.C. Chan, J.R. Grandis, Cross-talk between G protein-coupled receptor and epidermal growth factorreceptor signaling pathways contributes to growth and invasion of headand neck squamous cell carcinoma, Cancer Res. 66 (2006) 11831–11839.

[117] M. Scartozzi, I. Bearzi, R. Berardi, A. Mandolesi, C. Pierantoni, S. Cascinu,Epidermal growth factor receptor downstream signalling pathway inprimary colorectal tumours and related metastatic sites: optimising EGFR-targeted treatment options, Br. J. Cancer. 97 (2007) 92–97.

[118] M. Curtol, B.K. Cole, D. Lallemand, C.H. Liu, A.I. McClatchey, Contact-dependent inhibition of EGFR signaling by Nf2/Merlin, J. Cell Biol. 177(2007) 893–903.

[119] P.D. Schley, D.N. Brindley, C.J. Field, (n-3) PUFA alter raft lipidcomposition and decrease epidermal growth factor receptor levels in lipidrafts of human breast cancer cells, J. Nutr. 137 (2007) 548–553.

[120] H.Y. Oh, E.J. Lee, S. Yoon, B.H. Chung, K.S. Cho, S.J. Hong,Cholesterol level of lipid raft microdomains regulates apoptotic cell deathin prostate cancer cells through EGFR-mediated Akt and ERK signaltransduction, Prostate 67 (2007) 1061–1069.

[121] R.S. Misra, J.Q. Russell, A. Koenig, J.A. Hinshaw-Makepeace, R. Wen,D. Wang, H. Huo, D.R. Littman, U. Ferch, J. Ruland, M. Thome, R.C.Budd, Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation, J. Biol. Chem. 282 (2007)18374–19365.

[122] J. Zhong, J. Troppmair, U.R. Rapp, Independent control of cell survivalby Raf-1 and Bcl-2 at the mitochondria, Oncogene 20 (2001) 4807–4816.

[123] N. Khan, F. Afaq, M. Saleem, N. Ahmad, H. Mukhtar, Targeting multiplesignaling pathways by green tea polyphenol (–)-epigallocatechin-3-gallate, Cancer Res. 66 (2006) 2500–2505.

[124] R. Lu, X. Wang, Z.F. Chen, D.-F. Sun, X.-Q. Tian, J.-Y. Fang, Inhibitionof extracellular signal-regulated kinase/mitogen-activated protein kinasepathway decrease DNA-methylation in colon cancer cells, J. Biol Chem.282 (2007) 12249–12259.

[125] S.K. Patra, A. Patra, Lipid rafts in cancer metastasis — A workinghypothesis (Abstr.), 1st Ed, Proc. Am. Assoc. Cancer Res. (AACR), 44,2003, p. 64.

[126] L.E. Barrett, E.J. van Bockstaele, J.Y. Sul, H. Takano, P.G. Haydon, J.H.Eberwine, Elk-1 associates with the mitochondrial permeability transitionpore complex in neurons, Proc. Natl. Acad. Sci. 103 (2006) 5155–5160.

[127] L. Zhuang, J. Kim, R.M. Adam, K.R. Solomon, M.R. Freeman,Cholesterol targeting alters lipid raft composition and cell survival inprostate cancer cells and xenografts, J. Clin. Invest. 115 (2005) 959–968.

[128] C.C. Mastick, M.J. Brady, A.R. Saltiel, Insulin stimulates the tyrosinephosphorylation of caveolin, J. Cell Biol. 129 (1995) 1523–1531.

[129] J.S. Rao, Molecular mechanisms of glioma invasiveness: the role ofproteases, Nat. Rev., Cancer 3 (2003) 489–501.

[130] L.M. Coussens, Z. Werb, Inflammation and cancer. -Insight review.Nature 420 (2002) 860–867.

[131] L.A. Liotta, E.C. Khon, The microenvironment of the tumour–hostinterface—Insight review, Nature 411 (2001) 375–379.

[132] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000)57–70.

[133] K. Gaus, S. LeLay, N. Balasubramanian, M.A. Schwartz, Integrin-mediatedadhesion regulates membrane order, J. Cell Biol. 174 (2006) 725–734.

[134] E. Mira, R.A. Lacalle, J.M. Buesa, G.G. de Buitrago, S. Jimenez-Baranda, C. Gomez-Mouton, C. Martinez-A, S. Manes, Secreted MMP9promotes angiogenesis more efficiently than constitutively active MMP9bound to the tumour cell surface, J. Cell Sci. 117 (2004) 1847–1857.

[135] M.A. Schwartz, M.H. Ginsberg, Networks and crosstalk: integrinsignaling spreads, Nat. Cell Biol. 4 (2002) E65–E68.

[136] J.K. Slack-Davis, K.H. Martin, R.W. Tilghman, M. Iwanicki, E.J. Ung, C.Autry, M.J. Luzzio, B. Cooper, J.C. Kath, W.G. Roberts, J.T. Parsons,Cellular characterization of a novel focal adhesion kinase inhibitor,J. Biol. Chem. 282 (2007) 14845–14852.

[137] I.M. Ethell, D.W. Ethell, Matrix metalloproteinases in brain developmentand remodeling: synaptic functions and targets, J. Neurosci. Res. 85(2007) 2813–2823.

[138] J. Wolfers, A. Lozier, G. Raposo, A. Regnault, C. Thery, C. Masurier, C.Flament, S. Pouzieux, F. Faure, T. Tursz, E. Angevin, S. Amigorena, L.Zitvogel, Tumour-derived exosomes are a source of shared tumourrejection antigens for CTL cross-priming, Nat. Med. 7 (2001) 97–303.

[139] Y.S. Gho, H.K. Kleinman, G. Sosne, Angiogenic activity of human solubleintercellular adhesion molecule-1, Cancer Res. 59 (1999) 5128–5132.

[140] N. Sato, N. Maehara, G.H. Su, M. Goggins, Effects of 5-Aza-2′-deoxycytidine on matrix metalloproteinase expression and pancreaticcancer cell invasiveness, J. Natl. Cancer Inst. 95 (2003) 327–330.

[141] D.V. Rozanov, E.I. Deryugina, E.Z. Monosov, N.D. Marchenko, A.Y.Strongin, Aberrant, persistent inclusion into lipid rafts limits thetumourigenic function of membrane type-1 matrix metalloproteinase inmalignant cells, Exp. Cell Res. 293 (2004) 81–95.

[142] J. Jankun, S.H. Selman, R. Swiercz, E. Skrzypczak-Jankun, Whydrinking green tea could prevent cancer, Nature 387 (1997) 561.

[143] S.K. Mitra, D.A. Hanson, D.D. Schlaepfer, Focal adhesion kinase: incommand and control of cell motility, Nat. Rev., Mol. Cell Biol. 6 (2005)56–68.

[144] S. Abbi, J.L. Guan, Focal adhesion kinase: protein interactions andcellular functions, Histol. Histopathol. 17 (2002) 1163–1171.

[145] V. Gabarra-Niecko, M.D. Schaller, J.M. Dunty, FAK regulates biologicalprocesses important for the pathogenesis of cancer, Cancer MetastasisRev. 22 (2003) 359–374.

[146] G.W.McLean, N.O. Carragher, E.Avizienyte, J. Evans, V.G. Brunton,M.C.Frame, The role of focal-adhesion kinase in cancer — a new therapeuticopportunity, Nat. Rev., Cancer 5 (2005) 505–515.

[147] T. Babuke, R. Tikkanen, Dissecting the molecular function of reggie/flotillin proteins, Eur. J. Cell Biol. 86 (2007) 525–532.

[148] P. Hazarika, M.F. McCarty, V.G. Prieto, S. George, D. Babu, D. Koul, M.Bar-Eli, M. Duvic, Up-regulation of flotillin-2 is associated withmelanoma progression and modulates expression of the thrombin recep-tor protease activated receptor 1, Cancer Res. 64 (2004) 7361–7369.

[149] S.D. Doherty, V.G. Prieto, S.George, P. Hazarika,M.Duvic, High flotillin-2expression is associated with lymph node metastasis and Breslow depth inmelanoma, Melanoma Res. 16 (2006) 461–463.

[150] M. Boix-Chornet, M.F. Fraga, A. Villar-Garea, R. Caballero, J. Espada,A. Nunez, J. Casado, C. Largo, J.I. Casal, J.C. Cigudosa, L. Franco, M.Esteller, E. Ballester, Release of hypoacetylated and trymethylatedhistone H4 is an epigenetic marker of early apoptosis, J. Biol. Chem. 281(2006) 13540–135477.

[151] A.H. Boulares, A.G. Yakovlev, V. Ivanova, B.A. Stoica, G. Wang, S. Iyer,M. Smulson, Role of poly(ADP-ribose) polymerase (PARP) cleavage inapoptosis: caspase 3-resistant PARPmutant increases rates of apoptosis intransfected cells, J. Biol. Chem. 274 (1999) 22932–22940.

[152] N. Khan, F. Afaq, H. Mukhtar, Apoptosis by dietary factors: the suicidesolution for delaying cancer growth, Carcinogenesis 28 (2007) 233–239.

[153] C. Gajate, F. Mollinedo, Cytoskeleton-mediated death receptor and ligandconcentration in lipid rafts forms apoptosis-promoting clusters in cancerchemotherapy, J. Biol. Chem. 280 (2005) 11641–11647.

[154] C. Gajate, F. Mollinedo, The antitumor ether lipid ET-18-OCH(3) inducesapoptosis through translocation and capping of Fas/CD95 into membranerafts in human leukemic cells, Blood 98 (2001) 3860–3863.

[155] J.C. Reed, Drug insight: cancer therapy strategies based on restoration ofendogenous cell death mechanisms, Nat. Clin. Pract. Oncol. 3 (2006)388–398.

[156] S. Gurumurthy, K.M. Basudevan, V.M. Rangnekar, Regulation ofapoptosis in prostate cancer, Cancer Metastasis Rev. 20 (2001) 225–243.

[157] N. Itoh, S. Yonehara, A. Ishii, M. Yonehara, S.-I. Mizushima, M.Sameshima, A. Hase, Y. Seto, S. Nagata, The polypeptide encoded by thecDNA for human cell surface antigen Fas can mediate apoptosis, Cell 66(1991) 233–243.


[158] V.C. Canale, C.H. Smith, Chronic-lymphadenopathy simulating malig-nant lymphoma, J. Pediatr. 70 (1967) 891–899.

[159] J. Drappa, A.K. Vaishnaw, K.E. Sullivan, J.-L. Chu, K.B. Elkon, Fas genemutations in the Canale–Smith syndrome, an inherited lymphoprolifera-tive disorder associated with autoimmunity, New Engl. J. Med. 335(1996) 1643–1649.

[160] T. Brunner, R.J. Mogil, D. LaFace, N.J. Yoo, A. Mahboubi, F. Echeverri,S.J. Martin, W.R. Force, D.H. Lynch, C.F. Ware, D.R. Green, Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas, Nature 373 (1995) 441–444.

[161] S.-T. Ju, D.J. Panka, H. Cui, R. Ettinger, M. El-Khatib, D.H. Sherr, B.Z.Stanger, A. Marshak-Rothstein, Fas (CD95)/FasL interactions required forprogrammed cell death after T-cell activation, Nature 373 (1995) 444–448.

[162] J.B. Mannick, A. Hausladen, L. Liu, D.T. Hess, M. Zeng, Q.X. Miao, L.S.Kane, A.J. Gow, J.S. Stamler, Fas-induced caspase denitrosylation, Science284 (1999) 651–654.

[163] A.-O. Hueber, M. Zornig, D. Lyon, T. Suda, S. Nagata, G.I. Evan,Requirement for the CD95 receptor-ligand pathway in c-Myc-inducedapoptosis, Science 278 (1997) 1305–1309.

[164] A.-O. Hueber, CD95: more than just a death factor? Nat. Cell Biol. 2(2000) E23–E25.

[165] M.E. Peter, P.H. Krammer, The CD95(APO-1/Fas) DISC and beyond,Cell Death Differ. 10 (2003) 26–35.

[166] A. Lepple-Wienhues, C. Belka, T. Laun, A. Jekle, B. Walter, U. Wieland,M. Welz, L. Heil, J. Kun, G. Busch, M. Weller, M. Bamberg, E. Gulbins,F. Lang, Stimulation of CD95 (Fas) blocks T lymphocyte calciumchannels through sphingomyelinase and sphingolipids, Proc. Nat. Acad.Sci. 96 (1999) 13795–13800.

[167] H. Grassme, S. Kirschnek, J. Riethmueller, A. Riehle, G. von Kurthy, F.Lang,M.Weller, E. Gulbins, CD95/CD95 ligand interactions on epithelialcells in host defense to Pseudomonas aeruginosa, Science 290 (2000)527–530.

[168] P.L. Arscott, T. Stokes, A. Myc, T.J. Giordano, N.W. Thompson, J.R.Baker Jr., Fas (CD95) expression is up-regulated on papillary thyroidcarcinoma, J. Clin. Endocrinol. Metab. 84 (1999) 4246–4252.

[169] S.H. Lee, M.S. Shin, H.S. Kim,W.S. Park, S.Y. Kim, J.J. Jang, K.J. Rhim,J. Jang, H.K. Lee, J.Y. Park, R.R. Oh, S.Y. Han, J.H. Lee, J.Y. Lee, N.J.Yoo, Somatic mutations of Fas (Apo-1/CD95) gene in cutaneous squa-mous cell carcinoma arising from a burn scar, J. Invest. Dermatol. 114(1999) 122–126.

[170] X. Zhang, X. Miao, T. Sun, W. Tan, S. Qu, P. Xiong, Y. Zhou, D. Lin,Functional polymorphisms in cell death pathway genes FAS and FASLcontribute to the risk of lung cancer, J. Med. Genet. 42 (2005) 479–484.

[171] D.R. Green, T.A. Ferguson, The role of Fas ligand in immune privilege,Nat. Rev. Mol. Cell Biol. 2 (2001) 917–924.

[172] R. Watanabe-Fukunaga, C.I. Brannan, N.G. Copeland, N.A. Jenkins, S.Nagata, Lymphoproliferation disorder in mice explained by defects in Fasantigen that mediates apoptosis, Nature 356 (1992) 314–317.

[173] G. Frizzera, Y. Kaneko, M. Sakurai, Angioimmunoblastic lymphadeno-pathy and related disorders: a retrospective look in search of definitions,Leukemia 3 (1989) 1–5.

[174] J. Wu, T. Zhou, J. He, J.D. Mountz, Autoimmune disease in mice due tointegration of an endogenous retrovirus in an apoptosis gene, J. Exp.Med. 178 (1993) 461–468.

[175] A.Y. Savinov, A. Tcherepanov, E.A. Green, R.A. Flavell, A.V. Chervonsky,Contribution of Fas to diabetes development, Proc. Nat. Acad. Sci. 100(2003) 628–632.

[176] E. Song, S.-K. Lee, J. Wang, N. Ince, N. Ouyang, J. Min, J. Chen, P.Shankar, J. Lieberman, RNA interference targeting Fas protects micefrom fulminant hepatitis, Nat. Med. 9 (2003) 347–351.

[177] Y. Ma, H. Liu, H. Tu-Rapp, H.-J. Thiesen, S.M. Ibrahim, S.M. Cole, R.M.Pope, Fas ligation on macrophages enhances IL–1R1–Toll-like receptor4 signaling and promotes chronic inflammation, Nat. Immun. 5 (2004)380–387.

[178] A.M. Landau,K.C. Luk,M.-L. Jones, R. Siegrist-Johnstone, Y.K. Young, E.Kouassi, V.V. Rymar, A. Dagher, A.F. Sadikot, J. Desbarats, Defective Fasexpression exacerbates neurotoxicity in a model of Parkinson's disease,J. Exp. Med. 202 (2005) 575–581.

[179] G.H. Fisher, F.J. Rosenberg, S.E. Straus, J.K. Dale, L.A. Middelton, A.Y.Lin, W. Strober, M.J. Lenardo, J.M. Puck, Dominant interfering Fas genemutations impair apoptosis in a human autoimmune lymphoproliferativesyndrome, Cell 81 (1995) 935–946.

[180] M.C. Sneller, S.E. Straus, E.S. Jaffe, J.S. Jaffe, T.A. Fleisher, M. Stetler-Stevenson,W. Strober,A novel lymphoproliferative/autoimmune syndromeresembling murine lpr/gld disease, J. Clin. Invest. 90 (1992) 334–341.

[181] F. Rieux-Laucat, S. Blachere, S. Danielan, J.P. De Villartay, M. Oleastro,E. Solary, B. Bader-Meunier, P. Arkwright, C. Pondaré, F. Bernaudin, H.Chapel, S. Nielsen, M. Berrah, A. Fischer, F. Le Deist, Lymphoprolifera-tive syndrome with autoimmunity: a possible genetic basis for dominantexpression of the clinical manifestations, Blood 94 (1999) 2575–2582.

[182] A.I. Aspinall, A. Pinto, I.A. Auer, P. Bridges, J. Luider, L. Dimnik, K.D.Patel, K. Jorgenson, R.C. Woodman, Identification of new Fas mutationsin a patient with autoimmune lymphoproliferative syndrome (ALPS) andeosinophilia, Blood Cells Mol. Diseases 25 (1999) 227–238.

[183] E. Holzelova, C. Vonarbourg, M.-C. Stolzenberg, P.D. Arkwright, F. Selz,A.-M. Prieur, S. Blanche, J. Bartunkova, E. Vilmer, A. Fischer, F. LeDeist, F. Rieux-Laucat, Autoimmune lymphoproliferative syndrome withsomatic Fas mutations, New Engl. J. Med. 351 (2004) 1409–1418.

[184] R. Clementi, L. Dagna, U. Dianzani, L. Dupre, I. Dianzani, M. Ponzoni, A.Cometa, A. Chiocchetti,M.G. Sabbadini, C. Rugarli, F. Ciceri, R.Maccario,F. Locatelli, C. Danesino, M. Ferrarini, M. Bregni, Inherited perforin andFas mutations in a patient with autoimmune lymphoproliferative syndromeand lymphoma, New Engl. J. Med. 351 (2004) 1419–1424.

[185] A.K. Vaishnaw, J.R. Orlinick, J.-L. Chu, P.H. Krammer, M.V. Chao, K.B.Elkton, The molecular basis for apoptotic defects in patients with CD95(Fas/Apo-1) mutations, J. Clin. Invest. 103 (1999) 355–363.

[186] C.E. Jackson, R.E. Fischer, A.P. Hsu, S.M.Anderson, Y. Choi, J.Wang, J.K.Dale, T.A. Fleisher, L.A. Middelton, M.C. Sneller, M.J. Lenardo, S.E.Straus, J.M. Puck, Autoimmune lymphoproliferative syndrome with defec-tive Fas: genotype influences penetrance, Am. J. Hum. Genet. 64 (1999)1002–1014.

[187] D.A. Martin, L. Zheng, R.M. Siegel, B. Huang, G.H. Fisher, J. Wang, C.E.Jackson, J.M. Puck, J. Dale, S.E. Straus, M.E. Peter, P.H. Krammer, S.Fesik,M.J. Lenardo,DefectiveCD95/APO-1/Fas signal complex formationin the human autoimmune lymphoproliferative syndrome-type Ia, Proc.Natl. Acad. Sci. 96 (1999) 4552–4557 Note: Erratum (2004) Proc. Nat.Acad. Sci. 101, 7840.

[188] R.M. Siegel, J.K. Frederiksen, D.A. Zacharias, F.K.-M. Chan, M.Johnson, D. Lynch, R.Y. Tsien, M.J. Lenardo, Fas pre-association re-quired for apoptosis signaling and dominant inhibition by pathogenicmutations, Science 288 (2000) 2354–2357.

[189] N. Bidere, H.C. Su, M.J. Lenardo, Genetic disorders of programmed celldeath in the immune system, Annu. Rev. Immunol. 24 (2006) 321–352.

[190] O.V. Volpert, T. Zaichuk, W. Zhou, F. Reiher, T.A. Ferguson, P.M. Stuart,M. Amin, N.P. Bouck, Inducer-stimulated Fas targets activated endothe-lium for destruction by anti-angiogenic thrombospondin-1 and pigmentepithelium-derived factor, Nat. Med. 8 (2002) 349–357.

[191] P.R. Walker, T. Calzascia, V. Schnuriger, D. Chalmers, P. Saas, P.Y.Dietrich, Loss of Fas (CD95/APO-1) expression by antigen-specific cyto-toxic T cells is reversed by inhibiting DNA methylation, Cell. Immunol.206 (2000) 51–58.

[192] M. Shimizu, T. Yoshimoto, M. Sato, A. Matsuzawa, Y. Takeda, Fre-quency and resistance of CD95 (Fas/Apo-1) gene-transfected tumor cellsto CD95-mediated apoptosis by the elimination and methylation ofintegrated, DNA, Int. J. Cancer 119 (2006) 585–592.

[193] I. Petak, R.P. Danam, D.M. Tillman, R. Vernes, S.R. Howell, L. Berczi, L.Kopper, T.P. Brent, J.A. Houghton, Hypermethylation of the genepromoter and enhancer region can regulate Fas expression and sensitivityin colon carcinoma, Cell Death Differ. 10 (2003) 211–217.

[194] R. Castellano, B. Vire, M. Pion, V. Quivy, D. Olive, I. Hirsch, C. Van Lint,Y. Collette, Active transcription of the human FASL/CD95L/TNFSF6promoter region in T lymphocytes involves chromatin remodeling: role ofDNA methylation and protein acetylation suggest distinct mechanisms oftranscriptional repression, J. Biol. Chem. 281 (2007) 14719–14728.

[195] S. Hopkins-Donaldson, A. Ziegler, S. Kurtz, C. Bigosch, D. Kandioler, C.Ludwig, U. Zangemeister-Wittke, R. Stahel, Silencing of death receptor


and caspase-8 expression in small cell lung carcinoma cell lines andtumors by DNA methylation, Cell Death Differ. 10 (2003) 356–364.

[196] J. Peli, M. Schröter, C. Rudaz, M. Hahne, C. Meyer, E. Reichmann, J.Tschopp, Oncogenic Ras inhibits Fas ligand-mediated apoptosis bydownregulating the expression of Fas, EMBO J. 18 (1999) 1824–1831.

[197] M.M. van Noesel, S. van Bezouw, P.A. Voûte, J.G. Herman, R. Pieters, R.Versteeg, Clustering of hypermethylated genes in neuroblastoma, GenesChromosomes Cancer 38 (2003) 226–233.

[198] S. Santourlidis, U. Warskulat, A.R. Florl, S. Maas, T. Pulte, J. Fischer, W.Müller, W.A. Schulz, Hypermethylation of the tumor necrosis factor recep-tor superfamily 6 (APT1, Fas, CD95/Apo-1) gene promoter at rel/nuclearfactor kappaB sites in prostatic carcinoma,Mol. Carcinog. 32 (2001) 36–43.

[199] M.E. Peter, R.C. Budd, J. Desbarats, S.M. Hedrick, A.O. Hueber, M.K.Newell, L.B. Owen, R.M. Pope, J. Tschopp, H. Wajant, D. Wallach, R.H.Wiltrout, M. Zörnig, D.H. Lynch, The CD95 receptor: apoptosisrevisited, Cell 129 (2007) 447–450.

[200] K. Chakrabandhu, Z. Herincs, S. Huault, B. Dost, L. Peng, F. Conchonaud,D. Marguet, H.T. He, A.O. Hueber, Palmitoylation is required for efficientFas cell death signaling, EMBO J. 26 (2007) 209–220.

[201] K.H. Lee, C. Feing, V. Tchikov, R. Schickel, C. Hallas, S. Schutze, M.E.Peter, A.C. Chan, The role of receptor internalization in CD95 signaling,EMBO J. 25 (2006) 1009–1023.

[202] M. Miacznska, L. Pelkmans, M. Zerial, Not just a sink: endosomes incontrol of signal transduction, Curr. Opin. Cell Biol. 16 (2004) 400–406.

[203] K.L. Gould, A. Bretscher, F.S. Esch, T. Hunter, cDNA cloning andsequencing of the protein-tyrosine kinase substrate, ezrin, reveals homologyto band 4.1, EMBO J. 8 (1989) 4133–4142.

[204] R. Pakkanen, A. Vaheri, Cytovillin and other microvillar proteins ofhuman choriocarcinoma cells, J. Cell. Biochem. 41 (1989) 1–12.

[205] I. Saotome, M. Curto, A.I. McClatchey, Ezrin is essential for epithelialorganization and villus morphogenesis in the developing intestine, Dev.Cell 6 (2004) 855–864.

[206] S. Faure, L.I. Salazar-Fontana, M. Semichon, V.L.J. Tybulewicz, G.Bismuth, A. Trautmann, R.N. Germain, J. Delon, ERM proteins regulatecytoskeleton relaxation promoting T cell–APC conjugation, Nat. Immun.5 (2004) 272–279.

[207] A. Roumier, J.C. Olivo-Marin, M. Arpin, F. Michel, M.Martin, P.Mangeat,O. Acuto, A. Dautry-Varsat, A. Alcover, The membrane–microfilamentlinker ezrin is involved in the formation of the immunological synapse andin T cell activation, Immunity 15 (2001) 715–728.

[208] E.J. Allenspach, P. Cullinan, J. Tong, Q. Tang, A.G. Tesciuba, J.L.Cannon, S.M. Takahashi, R. Morgan, J.K. Burkhardt, A.I. Sperling,ERM-dependent movement of CD43 defines a novel protein complexdistal to the immunological synapse, Immunity 15 (2001) 739–750.

[209] O.-M.Mykkanen,M.Gronholm,M.Ronty,M.J. Lalowski, P. Salmikangas,H. Suila, O. Carpen, Characterization of human palladin, a microfilament-associated protein, Mol. Biol. Cell 12 (2001) 3060–3073.

[210] Y. Yu, J. Khan, C. Khanna, L. Helman, P.S. Meltzer, G. Merlino,Expression profiling identifies the cytoskeletal organizer ezrin and thedevelopmental homeoprotein Six-1 as key metastatic regulators, Nat.Med. 10 (2004) 175–181.

[211] R. Sharp, J.A. Recio, C. Jhappan, T. Otsuka, S. Liu, Y. Yu, W. Liu, M.Anver, F. Navid, L.J. Helman, R.A. DePinho, G. Merlino, Synergismbetween INK4a/ARF inactivation and aberrant HGF/SF signaling inrhabdomyosarcomagenesis, Nat. Med. 8 (2003) 1276–1280 Note: Cor-rigendum: 2003, Nat. Med. 9, 146. only.

[212] C. Khanna, X. Wan, S. Bose, R. Cassaday, O. Olomu, A. Mendoza, C.Yeung, R. Gorlick, S.M. Hewitt, L.J. Helman, The membrane–cyto-skeleton linker ezrin is necessary for osteosarcoma metastasis, Nat. Med.10 (2004) 182–186.

[213] P. Santana, L.A. Pena, A. Haimovitz-riedman, S. Martin, D. Green, M.McLoughlin, C. Cordon-Cardo, E.H. Schuchman, Z. Fuks, R. Kolesnick,Acid sphingomyelinase-deficient human lymphoblasts and mice aredefective in radiation-induced apoptosis, Cell 86 (1996) 189–199.

[214] M. Suchi, T. Dinur, R.J. Desnick, S. Gatt, L. Pereira, E. Gilboa, E.H.Schuchman, Retroviral-mediated transfer of the human acid sphingomye-linase cDNA: correction of the metabolic defect in cultured Niemann–Pickdisease cells, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 3227–3231.

[215] B. Otterbach, W. Stoffel, Acid sphingomyelinase-deficient mice mimic theneurovisceral form of human lysosomal storage disease (Niemann–Pickdisease), Cell 81 (1995) 1053–1061.

[216] K. Horinouchi, S. Erlich, D.P. Perl, K. Ferlinz, C.L. Bisgaier, K. Sandhoff,R.J. Desnick, C.L. Stewart, E.H. Schuchman, Acid sphingomyelinasedeficient mice: a model of types A and B Niemann–Pick disease, Nat.Genet. 10 (1995) 288–293.

[217] H. Grassme, E. Gulbins, B. Brenner, K. Ferlinz, K. Sandhoff, K. Harzer, F.Lang, T.F. Meyer, Acidic sphingomyelinase mediates entry of N. gonor-rhoeae in nonphagocytic cells. Cell 91 (1997) 605–615.

[218] W. Malorni, A.M. Giammarioli, T. Garofalo, M. Sorice, Dynamics oflipid raft components during lymphocyte apoptosis: the paradigmatic roleof GD3, Apoptosis 12 (2007) 941–949.

[219] A.E. Cremesti, F.M. Goni, R. Kolesnick, Role of sphingomyelinase andceramide in modulating rafts: do biophysical properties determinebiologic outcome? FEBS Lett. 531 (2002) 47–53.

[220] R. Kolesnick, The therapeutic potential of modulating the ceramide/sphingomyelin pathway, J. Clin. Invest. 110 (2002) 3–8.

[221] A. Morales, H. Lee, F.M. Goni, R. Kolesnick, J.C. Fernandez-Checa,Sphingolipids and cell death, Apoptosis 12 (2007) 923–939.

[222] S. DeMorrow, S. Glaser, H. Francis, J. Venter, B. Vaculin, S. Vaculin, G.Alpini, Opposing actions of endocannabinoids on cholangiocarcinomagrowth: recruitment of Fas and Fas ligand to lipid rafts, J. Biol. Chem.282 (2007) 13098–13113.

[223] W. Elyassaki, S. Wu, Lipid rafts mediate ultraviolet light-induced Fasaggregation in M624 melanoma cells, Photochem. Photobiol. 82 (2006)787–792.

[224] M.Miyaji, Z.X. Jin, S. Yamaoka, R. Amakawa, S. Fukuhara, S.B. Sato, T.Kobayashi, N. Domae, T. Mimori, E.T. Bloom, T. Okazaki, H. Umehara,Role of membrane sphingomyelin and ceramide in platform formation forFas-mediated apoptosis, J. Exp. Med. 202 (2005) 249–259.

[225] F. Mollinedo, C. Gajate, Fas/CD95 death receptor and lipid rafts: newtargets for apoptosis-directed cancer therapy, Drug Resist. Updat. 9 (2006)51–73.

[226] C. Gajate, E. Del Canto-Janez, A.U. Acuna, F. Amat-Guerri, E. Geijo, A.M.Santos-Beneit, R.J. Veldman, F. Mollinedo, Intracellular triggering of Fasaggregation and recruitment of apoptotic molecules into Fas enriched raftsin selective tumor cell apoptosis, J. Exp. Med. 200 (2004) 353–365.

[227] C. Gajate, F.Mollinedo, Edelfosine and perifosine induce selective apoptosisin multiple myeloma by recruitment of death receptors and downstreamsignaling molecules into lipid rafts, Blood 109 (2007) 711–719.

[228] C. Gajate, A.M. Santos-Beneit, A. Macho, M. Lazaro, A. Hernandez-DeRojas, M. Modolell, E. Muñoz, F. Mollinedo, Involvement of mito-chondria and caspase-3 in ET-18-OCH(3)-induced apoptosis of humanleukemic cells, Int. J. Cancer. 86 (2000) 208–218.

[229] W.J. van Blitterswijk, A.H. van Der Luit, R.J. Veldman, M. Verheij, J.Borst, Ceramide: second messenger or modulator of membrane structureand dynamics? Biochem. J. 369 (2003) 199–211.

[230] T.A. Nurminen, J.M. Holopainen, H. Zhao, P.K. Kinnunen, Observationof topical catalysis by sphingomyelinase coupled to microspheres, J. Am.Chem. Soc. 124 (2002) 12129–12134.

[231] H.W. Huang, E.M. Goldberg, R. Zidovetzki, Ceramides modulate proteinkinase C activity and perturb the structure of phosphatidylcholine/phos-phatidylserine bilayers, Biophys. J. 77 (1999) 1489–1497.

[232] M.P. Veiga, J.L.R. Arrondo, F.M. Goni, A. Alonso, D. Marsh, Interactionof cholesterol with sphingomyelin in mixed membranes containingphosphatidylcholine, studied by spin-label ESR and IR spectroscopies. Apossible stabilization of gel-phase sphingolipid domains by cholesterol,Biochemistry 40 (2001) 2614–2622.

[233] C. Yu, M. Alterman, R.T. Dobrowsky, Ceramide displaces cholesterolfrom lipid rafts and decreases the association of the cholesterol bindingprotein caveolin-1, J. Lipid Res. 46 (2005) 1678–1691.

[234] K. Simons, E. Ikonen, How cells handle cholesterol, Science 290 (2000)1721–1726.

[235] R.M. White, On the occurrence of crystals in tumours, J. Pathol. Bac-teriol. 13 (1909) 3–10.

[236] M. Yasuda, Lipid metabolism of tumours, Proc. Soc. Exp. Biol. Med. 27(1930) 944–945.


[237] G.M.I. Swyer, The cholesterol content of normal and enlarged prostates,Cancer Res. 2 (1942) 372–375.

[238] M.R. Freeman, K.R. Solomon, Cholesterol and prostate cancer, J. CellBiochem. 91 (2004) 54–69.

[239] Y. Hamon, C. Broccardo, O. Chambenoit, M.F. Luciani, F. Toti, S. Chaslin,J.M. Freyssinet, P.F. Devaux, J. McNeish, D. Marguet, G. Chimini, ABC1promotes engulfment of apoptotic cells and transbilayer redistribution ofphosphatidylserine, Nat Cell Biol. 2 (2000) 399–406.

[240] E. Orsó, C. Broccardo, W.E. Kaminski, A. Böttcher, G. Liebisch, W.Drobnik, A. Götz, O. Chambenoit, W. Diederich, T. Langmann, T.Spruss, M.F. Luciani, G. Rothe, K.J. Lackner, G. Chimini, G. Schmitz,Transport of lipids from Golgi to plasma membrane is defective in tangierdisease patients and Abc1-deficient mice, Nat Genet. 24 (2000) 192–196.

[241] R.L. Gebhard, R.V. Clayman, W.F. Prigge, R. Figenshau, N.A. Staley, C.Reesey, A. Bear, Abnormal cholesterol metabolism in renal clear cellcarcinoma, J. Lipid Res. 28 (1987) 1177–1184.

[242] M.P. Lerner, S.W. Lucid, G.J. Wen, R.E. Nordquist, Selected areamembrane shedding by tumour cells, Cancer Lett. 20 (1983) 125–130.

[243] A. Le Bivic, H. Sari, M. Reynier, S. Lebec, F. Bardin, Differences in lipidcharacteristics of autologous human melanoma cell lines with distinctbiological properties, J. Natl. Cancer Inst. 79 (1987) 1181–1188.

[244] R. Dahiya, B. Boyle, B.C. Goldberg,W.H. Yoon, B. Knoety, K. Chen, T.S.Yen, W. Blumenfeld, P. Narayan, Metastasis associated alteration inphospholipids and fatty acids of human prostatic adenocarcinoma celllines, Biochem. Cell. Biol. 70 (1992) 548–554.

[245] W.J. van Blitterswijk, G. de Veer, J.H. Krol, P. Emmelot, Comparativeanalysis of purified plasma membranes and shed extracellular membranevesicles from normal murine thymocytes and leukemic GRSL cells,Biochim. Biophys. Acta 688 (1982) 495–504.

[246] D.D. Poutsiaka, D.D. Taylor, E.M. Levy, P.H. Black, Inhibition ofrecombinant interferon-γ-induced Ia antigen expression by shed B16F10melanoma cell membrane vesicles, J. Immunol. 134 (1985) 145–150.

[247] J. Wolfers, A. Lozier, G. Raposo, A. Regnault, C. Thery, C. Masurier, C.Flament, S. Pouzieux, F. Faure, T. Tursz, E. Angevin, S. Amigorena, L.Zitvogel, Tumour-derived exosomes are a sourceof shared tumour rejectionantigens for CTL cross-priming, Nat. Med. 7 (2001) 297–303.

[248] A. Ginestra, S. Monia, G. Seghezzi, V. Dolo, H. Nagase, P. Mignatti,M.L. Vittorelli, Urokinase plasminogen activator and gelatinases areassociated with membrane vesicles shed by human HT1080 fibrosar-coma cells, J. Biol. Chem. 272 (1997) 17216–17222.

[249] V. Dolo, A. Ginestra, D. Cassara, S. Violini, G. Luciania, G. Torrisi, H.Nagase, S. Canevari, A. Pavan, M.L. Vittorelli, Selective localization ofmatrix metalloproteinase 9, β1 integrins and human lymphocyte antigenclass I molecules on membrane vesicles shed by 8701-BC breastcarcinoma cells, Cancer Res. 58 (1998) 4468–4474.

[250] C. Chetty, P. Bhoopathi, S.S. Lakka, J.S. Rao, in press. MMP-2 siRNAinduced Fas/CD95-mediated extrinsic II apoptotic pathway in the A549lung adenocarcinoma cell line. Oncogene.

[251] B.C. Barnhart, P. Legembre, E. Pietras, C. Bubici, G. Franzoso, M.E. Peter,CD95 ligand induces motility and invasiveness of apoptosis-resistant tumorcells, EMBO J. 23 (2004) 3175–3185.

[252] M.Watabe, K. Hishikawa, A. Takayanagi, N. Shimizu, T. Nakaki, Caffeicacid phenethyl ester induces apoptosis by inhibition of NFκB andactivation of Fas in human breast cancer MCF-7 cells, J. Biol. Chem. 279(2004) 6017–6026.

[253] Y. Guo, P. Pakneshan, J. Gladu, A. Slak, M. Szyf, S.A. Rabbani, Regulationof DNA methylation in human breast cancer: effect on the urokinase-typeplasminogen activator gene production and tumour invasion, J. Biol. Chem.277 (2002) 41571–41579.

[254] S.M. Pulukuri, N. Estes, J. Patel, J.S. Rao, Demethylation-linkedactivation of urokinase plasminogen activator is involved in progressionof prostate cancer, Cancer Res. 67 (2007) 930–939.

[255] P. Pakneshan, M. Szyf, R. Farias-Eisner, S.A. Rabbani, Reversal of thehypomethylation status of urokinase (uPA) promoter blocks breast cancergrowth and metastasis, J. Biol. Chem. 279 (2004) 31735–31744.

[256] A. Ostapkowicz, K. Inai, L. Smith, S. Kreda, J. Spychala, Lipid raftsremodeling in estrogen receptor-negative breast cancer is reversed byhistone deacetylase inhibitor, Mol. Cancer Ther. 5 (2006) 238–245.

[257] S.K. Patra, A. Patra, R. Dahiya, Histone deacetylase and DNA methyl-transferase in human prostate cancer, Biochem. Biophys. Res. Commun.287 (2001) 705–713.

[258] D.A. Stewart, C.R. Cooper, R.A. Sikes, Changes in extracellular matrix(ECM) and ECM-ssociated proteins in the metastatic progression ofprostate cancer, Reprod. Biol. Endocrinol. 2 (2004) 2.

[259] M. Kanzaki, J.E. Pessin, Caveolin associated filamentous actin (cav-actin)defines a novel F-actin structure in adipocyte, J. Biol. Chem. 277 (2002)25867–25869.

[260] I.A. Prior, A. Harding, J. Yan, J. Sluimer, R.G. Parton, J.F. Hancock,GTP-dependent segregation of H-ras from lipid rafts is required forbiological activity, Nat. Cell Biol. 3 (2001) 368–375.

[261] I.A. Prior, C. Muncke, R.G. Parton, J.F. Hancock, Direct visualization ofRas proteins in spatially distinct cell surface microdomains, J. Cell Biol.160 (2003) 165–170.

[262] Y.C. Li, M.J. Park, S.-K. Ye, C.-W. Kim, Y.-N. Kim, Elevated levels ofcholesterol-rich lipid rafts in cancer cells are correlated with apoptosissensitivity induced by cholesterol depleting agents, Am. J. Pathol. 168(2006) 1107–1118.

[263] H. Shimamura, Y. Terada, T. Okado, H. Tanaka, S. Inoshita, S. Sasaki,The PI3-kinase-Akt pathway promotes mesangial cell survival and in-hibits apoptosis in vitro via NF-κB and Bad, J. Am. Soc. Nephrol. 14(2003) 1427–1434.

[264] J.A. Romashkova, S.S. Makarov, NF-κB is a target of AKT in anti-apoptotic PDGF signaling, Nature 401 (1999) 86–90.

[265] D.J. Panka, T. Mano, T. Suhara, K. Walsh, J.W. Mier, Phosphatidylino-sitol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells,J. Biol. Chem. 276 (2001) 6893–6896.

[266] S.R. Datta, H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, M.E.Greenberg, Akt phosphorylation of BAD couples survival signals to thecell-intrinsic death machinery, Cell 91 (1997) 231–241.

[267] L.P. Kane, V.S. Shapiro, D. Stokoe, A. Weiss, Induction of NF-κB by theAkt/PKB kinase, Curr. Biol. 9 (1999) 601–604.

[268] O. Micheau, S. Lens, O. Gaide, K. Alevizopoulos, J. Tschopp, NF-κB signalsinduce the expression of c-FLIP, Mol. Cell. Biol. 21 (2001) 5299–5305.

[269] C. Chen, L.C. Edelstein, C. Gelinas, The Rel/NF-kappaB family directlyactivates expression of the apoptosis inhibitor Bcl-x(L), Mol, Cell Biol.20 (2000) 2687–2695.

[270] C. Partovian, M. Simons, Regulation of protein kinase B/Akt activity andSer473 phosphorylation by protein kinase C-alpha in endothelial cells,Cell. Signal. 16 (2004) 951–957.

[271] S. Elhyany, E. Assa-Kunik, S. Tsory, T. Muller, S. Fedida, S. Segal, D.Fishman, The integrity of cholesterol-enriched microdomains is essentialfor the constitutive high activity of protein kinase B in tumour cells,Biochem. Soc. Trans. 32 (2004) 837–839.

[272] M.M. Hill, J. Feng, B.A. Hemmings, Identification of a plasmamembrane raft-associated PKB Ser473 kinase activity that is distinctfrom ILK and PDK1, Curr. Biol. 12 (2002) 1251–1255.

[273] X. Chen, M.D. Resh, Cholesterol depletion from the plasma membranetriggers ligand-independent activation of the epidermal growth factorreceptor, J. Biol. Chem. 277 (2002) 49631–49637.

[274] C. Mineo, G.N. Gill, R.G. Anderson, Regulated migration of epider-mal growth factor receptor from caveolae, J. Biol. Chem. 274 (1999)30636–30643.

[275] J. Grossmann,Molecular mechanisms of “detachment-induced apoptosis-Anoikis”, Apoptosis 7 (2002) 247–260.

[276] P. Lajoie, E.A. Partridge, G. Guay, J.G. Goetz, J. Pawling, A. Lagana, B.Joshi, J.W. Dennis, I.R. Nabi, Plasma membrane domain organizationregulates EGFR signaling in tumor cells, J. Cell Biol. 179 (2007) 341–356.

[277] D.Mandal, A.Majumder, P. Das, M. Kundu, J. Basu, Fas-, caspase 8-, andcaspase 3-dependent signaling regulates the activity of the aminopho-spholipid translocase and phosphatidylserine externalization in humanerythrocytes, J. Biol. Chem. 280 (2005) 39460–39467.

[278] S. Adachi, T. Nagao, H.I. Ingolfosson, F.R. Maxfield, O.S. Andersen, L.Kopelovich, I.B. Weinstein, The inhibitory effect of (−)-epigallocatechingallate on activation of the epidermal growth factor receptor is associatedwith altered lipid order in HT29 colon cancer cells, Cancer Res. 67 (2007)6493–6501.

Review Dissecting lipid raft facilitated cell signaling ...€¦ · Review Dissecting lipid raft...

Documents

Transcript of Review Dissecting lipid raft facilitated cell signaling ...€¦ · Review Dissecting lipid raft...